Protein Kinase Catalytic Subunit Research Articles

Molecular determinants and signaling effects of PKA RIα phase separation

Spatiotemporal regulation of intracellular signaling molecules, such as the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), ensures proper cellular function. Liquid-liquid phase separation (LLPS) of the ubiquitous PKA regulatory subunit RIα promotes cAMP compartmentation and signaling specificity. However, the molecular determinants of RIα LLPS remain unclear. Here, we reveal that two separate dimerization interfaces, combined with the cAMP-induced unleashing of the PKA catalytic subunit (PKA-C) from the pseudosubstrate inhibitory sequence, drive RIα condensate formation in the cytosol of mammalian cells, which is antagonized by docking to A-kinase anchoring proteins. Strikingly, we find that the RIα pseudosubstrate region is critically involved in forming a non-canonical R:C complex, which recruits active PKA-C to RIα condensates to maintain low basal PKA activity in the cytosol. Our results suggest that RIα LLPS not only facilitates cAMP compartmentation but also spatially restrains active PKA-C, thus highlighting the functional versatility of biomolecular condensates in driving signaling specificity.

Abstract 137: Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma

Abstract Tumor cells of almost all patients with fibrolamellar carcinoma (FLC) have a somatic mutation, a ~400 kB deletion on one copy of chromosome 19 that results in a fusion transcript of DNAJB1 to PRKACA, the catalytic subunit of protein kinase A. The resulting fusion oncoprotein, DNAJB1::PRKACA, has kinase activity, and is required and sufficient for transformation. To understand the oncogenesis of this tumor and to help identify potential therapeutic targets, we explored the pathways of pathogenesis mediated by the aberrant kinase. We explored three mechanisms that might explain transformation by the oncogenic kinase: - A difference in the substrate specificity of DNAJB1::PRKACA kinase versus native PRKACA kinase. - A difference in the interactome of DNAJB1::PRKACA kinase versus native PRKACA kinase. - An increase of total catalytic kinase activity (DNAJB1::PRKACA + PRKACA) versus the native PRKACA condition. We found, comparing cytosolic extracts of tumor and normal liver cells, that there are differences in the substrate specificity of the DNAJB1::PRKACA kinase. Additionally, such comparisons revealed differences in the interactions between the catalytic subunit of the kinase and cytosolic components. We also demonstrated a consistent increase in total PRKACA catalytic subunit in the FLC tumor, relative to the adjacent nontransformed tissue. Further, there is an increase in free, unregulated catalytic activity in the tumor cells and a change in the localization of the catalytic subunit in the tumor cells. Additionally, we have found that this increase in free catalytic activity is sufficient to account for the changes in the transcriptome in tumor cells of FLC patients. Importantly, enhanced expression in hepatocytes of either DNAJB1::PRKACA kinase or native PRKACA kinase caused very similar changes in the transcriptome, and the resultant transcriptomes closely resembled that of tumor cells from FLC patients. This suggests that the DNAJB1 domain is dispensable and not critical for transformation. In our investigation of patient tumors we found two rare and different subclasses of FLC that are indistinguishable from “classic” FLC, based on their transcriptome and based on their drug response profiles. Importantly, both subclasses are associated with changes of PRKACA, but neither involves the DNAJB1 domain. Thus, the DNAJB1 domain is not a critical component of transformation, and the significant driver is an increase of the cellular catalytic activity. Citation Format: Mahsa Shirani, Michael Tomasini, Solomon Levin, soeren heissel, Henrik Molina, Ype De Jong, charles Rice, Philip Coffino, Barbara Lyons, Sanford Simon. Increased activity of protein kinase A is sufficient to cause fibrolamellar carcinoma [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 137.

Glutamine antagonist DRP-104 in combination with durvalumab in patients with advanced fibrolamellar carcinoma (FLC) following progression on prior anti-PD(L)1 therapy.

TPS573 Background: Fibrolamellar carcinoma (FLC) is a rare, aggressive form of primary liver cancer predominantly affecting children and young adults under the age of 30. There is no established standard therapy and most patients with advanced disease succumb to their illness with a median prognosis of only 12 months. A chimeric transcript between DNAJB1 and PRKACA, which encodes the catalytic subunit of protein kinase A (PKA), was identified as a signature genomic event in FLC. However, pharmacological inhibition of PKAc for FLC with traditional small molecule inhibitors has been infeasible due to unacceptable on-target toxicity. Preclinical work from our laboratory and others has revealed that the DNAJB1- PRKACA fusion results in a metabolic rewiring of the tumor characterized by glutamine dependence. This glutamine dependence also creates a nutrient-depleted tumor immune microenvironment (TiME) enriched in immunosuppressive metabolites (e.g., ammonia, acidosis) that prohibits an effective antitumor immune response. Through this trial, we will test the hypothesis that glutamine antagonism in FLC reverses resistance to immune checkpoint inhibitor (ICI) therapy through modulating the TiME. Methods: We are conducting a single-arm phase 1b/2 clinical trial of glutamine antagonist DRP-104 in combination with durvalumab in children (age ≥ 12) and adults with advanced FLC who have progressed on prior anti-PD(L)1 therapy. Patients will receive DRP-104 (145 mg s.c. twice weekly) in combination with a fixed dose of durvalumab (1500 mg i.v. every 28 days). The primary clinical endpoints are safety and objective response rate (ORR), defined as the percentage of patients achieving a complete response (CR) or partial response (PR) based on the Response Evaluation Criteria in Solid Tumors (RECIST 1.1). Secondary objectives include progression free survival (PFS), overall survival (OS), and immunological correlates. Key eligibility criteria include histologically confirmed FLC that is metastatic or unresectable; demonstrated radiographic progression on at least one line of prior anti-PD(L)1 therapy; Age ≥ 12 years; ECOG performance status of ≤2; measurable disease according to RECIST 1.1; and adequate end organ function. Patients will undergo fresh core tumor biopsy pre-treatment and at approximately week 4. Biopsies will be used to prepare paraffin embedded and flash-frozen samples. Research bloodwork will be collected prior to dosing at baseline, prior to dosing on cycles 2-5, and every 3 months thereafter to be processed for PBMC isolation. This study has been registered under NCT06027086 and is expected to begin enrollment in December 2023. Clinical trial information: NCT06027086 .

Abstract C057: Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways

Abstract Background: Fibrolamellar carcinoma (FLC) is a primary liver cancer, which arises in a background of normal liver. The DNAJB1-PRKACA (DP) fusion gene is present in the majority of FLC and is an oncogenic driver in animal models. The oncogenic mechanism of DP is unclear. To date, transcriptomic analyses of FLC have been based on bulk RNA sequencing. Given the lack of cell-specific data in bulk transcriptomic analysis, we hypothesize that using single nucleus RNA sequencing (snRNA-Seq) will allow for identification of significantly dysregulated signaling pathways specific to tumor cells. Methods: Single nuclei were isolated from 7 FLC tumors and 4 normal liver (NL). The Chromium Next GEM Single Cell 3’ Reagent Kit was used to generate sequencing libraries. All samples passing QC were submitted for sequencing by GeneWiz with a read depth of 20,000 reads/cell. An R-based pipeline for the single-cell profiling was built using Seurat R toolkit. Cells exhibiting extremely low or high library sizes, gene features outside the 95% confidence interval, or displaying high mitochondrial content were filtered out. Clusters from snRNA-Seq analysis were annotated using singleR, enrichR, and publicly available datasets. Results: FLC and NL nuclei counts were 98664 and 56644, respectively. We generated thirty one clusters and annotated 21 cell types. The FLC tumor group was annotated based on previously published FLC bulk transcriptomic data. We identified increased expression of SLC16A14, TMEM163, PDE10A, OAT, and GALNTL6 in FLC tumor cells. The predominant cells types and percentage of cells (%NL/%FLC) per group were: hepatocytes (43/57), endothelial cells (18/82), FLC tumor cells (15/85), stellate cells (28/72), Kupffer cells (50/50), cholangiocytes (62/38), Other 1 (22/78), T cells (36/64), and fibroblasts (69/31). FLC tissue contained only 3% of the liver sinusoidal endothelial cells. Comparison of FLC tumor cells demonstrated differential upregulation of 1879 and downregulation of 1579 genes in tumor vs. NL. Gene ontology (GO) enrichment identified the following top processes in FLC tumor cells: ATP metabolic process, cellular respiration, and response to peptide hormone. GO analysis of hepatocytes identified: ribonucleotide metabolic process, energy derivation by oxidation of organic compounds, and response to peptide hormone as significant pathways. Conclusions: FLC tumor cells demonstrate alterations in mitochondrial processes and energetics. FLC cells are known to contain abundant mitochondria, which is not observed in other primary liver epithelial cancers. The PRKACA component of DP fusion protein is the catalytic subunit of protein kinase A (PKA). Since PKA is known to regulate mitochondrial function, these data support the possibility that DP may directly alter mitochondrial function. Another interesting finding is the presence of the FLC tumor cell type in NL group (15%), which may suggest its dedifferentiation status or its cell-of-origin as a subpopulation of liver cells distinct from mature hepatocytes or cholangiocytes. Citation Format: Nihal Bharath, Biju Isaac, Marc S. Sherman, Colton J. Smith, Michael Karski, Liang Sun, Brian J. Pepe-Mooney, Ina Kycia, Michael Rogers, Wolfram Goessling, Khashayar Vakili. Fibrolamellar carcinoma single-nucleus RNA sequencing reveals alteration of mitochondrial energetic pathways [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C057.

Retraction Note: Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins.

Retraction Note: Protein kinase catalytic subunit (PKAcat) from bovine lens: purification, characterization and phosphorylation of lens crystallins.

GalNAc-conjugated siRNA targeting the DNAJB1-PRKACA fusion junction in fibrolamellar hepatocellular carcinoma

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models invitro and invivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.

Redox Modification of PKA-Cα Differentially Affects Its Substrate Selection.

The cyclic AMP-dependent protein kinase (PKA) plays an essential role in the regulation of many important cellular processes and is dysregulated in several pervasive diseases, including diabetes, cardiovascular disease, and various neurodegenerative disorders. Previous studies suggest that the alpha isoform of the catalytic subunit of PKA (PKA-Cα) is oxidized on C199, both in vitro and in situ. However, the molecular consequences of these modifications on PKA-Cα's substrate selection remain largely unexplored. C199 is located on the P + 1 loop within PKA-Cα's active site, suggesting that redox modification may affect its kinase activity. Given the proximity of C199 to the substrate binding pocket, we hypothesized that oxidation could differentially alter PKA-Cα's activity toward its substrates. To this end, we examined the effects of diamide- and H2O2-dependent oxidation on PKA-Cα's activity toward select peptide and protein substrates using a combination of biochemical (i.e., trans-phosphorylation assays and steady-state kinetics analysis) and biophysical (i.e., surface plasmon resonance and fluorescence polarization assays) strategies. These studies suggest that redox modification of PKA-Cα differentially affects its activity toward different substrates. For instance, we found that diamide-mediated oxidation caused a marked decrease in PKA-Cα's activity toward some substrates (e.g., Kemptide and CREBtide) while having little effect on others (e.g., Crosstide). In contrast, H2O2-dependent oxidation of PKA-Cα led to an increase in its activity toward each of the substrates at relatively low H2O2 concentrations, with differential effects at higher peroxide concentrations. Together, these studies offer novel insights into crosstalk between redox- and phosphorylation-dependent signaling pathways mediated by PKA. Likewise, since C199 is highly conserved among AGC kinase family members, they also lay the foundation for future studies designed to elucidate the role of redox-dependent modification of kinase substrate selection in physiological and pathological states.

Edmond Fischer's kinase legacy: History of the protein kinase inhibitor and protein kinaseA.

Although Fischer's extraordinary career came to focus mostly on the protein phosphatases, after his co-discovery of Phosphorylase Kinase with Ed Krebs he was clearly intrigued not only by cAMP-dependent protein kinase (PKA), but also by the heat-stable, high-affinity protein kinase inhibitor (PKI). PKI is an intrinsically disordered protein that contains at its N-terminus a pseudo-substrate motif that binds synergistically and with high-affinity to the PKA catalytic (C) subunit. The sequencing and characterization of this inhibitor peptide (IP20) were validated by the structure of the PKA C-subunit solved first as a binary complex with IP20 and then as a ternary complex with ATP and two magnesium ions. A second motif, nuclear export signal (NES), was later discovered in PKI. Both motifs correspond to amphipathic helices that convey high-affinity binding. The dynamic features of full-length PKI, recently captured by NMR, confirmed that the IP20 motif becomes dynamically and sequentially ordered only in the presence of the C-subunit. The type I PKA regulatory (R) subunits also contain a pseudo-substrate ATPMg2-dependent high-affinity inhibitor sequence. PKI and PKA, especially the Cβ subunit, are highly expressed in the brain, and PKI expression is also cell cycle-dependent. In addition, PKI is now linked to several cancers. The full biological importance of PKI and PKA signaling in the brain, and their importance in cancer thus remains to be elucidated.

DNA‑PKcs phosphorylation specific inhibitor, NU7441, enhances the radiosensitivity of clinically relevant radioresistant oral squamous cell carcinoma cells.

Radioresistant cancer cells lead to poor prognosis after radiotherapy. However, the mechanisms underlying cancer cell radioresistance have not been fully elucidated. Thus, the DNA damage response of clinically relevant radioresistant oral squamous cell carcinoma HSC2-R cells, established by long-term exposure of parental HSC2 cells to fractionated radiation, was investigated. The DNA double-strand break (DSB) repair protein-specific inhibitor, NU7441, which targets DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation, and IBR2, which targets Rad51, were administered to HSC2 and HSC2-R cells. NU7441 administration eliminated colony formation in both cell lines under 6 Gy X-ray irradiation, whereas IBR2 did not affect colony formation. NU7441 and IBR2 significantly enhanced 6 Gy X-ray irradiation-induced apoptosis in HSC2-R cells. In HSC2-R cells, cell cycle arrest released earlier than in HSC2 cells, and phosphorylated-H2A histone family member X (γH2AX) expression rapidly decreased. Following NU7441 administration, γH2AX expression and the cell percentages of the G2/M phase were not decreased at 48 h after treatment in HSC2-R cells. DNA-PKcs has been demonstrated to regulate non-homologous end-joining (NHEJ) and homologous recombination (HR) repair, and the later phase of DSB repair is dominated by HR. Therefore, the results of the present study indicated that the DSB repair mechanism in HSC2-R cells strongly depends on NHEJ and loss of HR repair function. The present study revealed a potential mechanism underlying the acquired radioresistance and therapeutic targets in radioresistant cancer cells.

Very Low Protein Diets Induce a Rapid Decrease in Hepatic cAMP-Dependent Protein Kinase Followed by a Slower Increase in Adenylyl Cyclase Activity in Rats

Recent evidence indicates that cAMP-mediated responses are desensitized in liver during malnutrition. While receptor-stimulated production of cAMP is increased in hepatocytes from rats fed very low protein diets for 14 d, activity of cAMP-dependent protein kinase (PKA) is decreased in liver cytosol. The present study investigated the time course for this desensitization. Weanling rats were fed either a 0.5 (malnourished) or 15% protein (control) diet for 1, 3, 7 or 14 d. Total PKA activity decreased after only 3 d of feeding the low protein diet. This decrease was confined to the cytosolic compartment and was associated with a lower quantity of immunoreactive RI regulatory subunit of PKA, with no difference in the quantity of immunoreactive RII regulatory subunit. In contrast, basal-, MnCl2- and guanine nucleotide regulatory protein-stimulated adenylyl cyclase activities were not greater in liver membranes of malnourished rats than in those of the control rats until the 2nd wk of feeding. Greater activity was paralleled by an increase in the quantity of the stimulatory guanine nucleotide regulatory protein at d 14. The inhibitory guanine nucleotide regulatory protein quantity did not differ between dietary groups. Greater cAMP production was not mediated by changes in PKA phosphorylation of adenylyl cyclase because preincubation of membranes with purified PKA catalytic subunit decreased MnCl2-stimulated cAMP production equally in liver membranes of both control and malnourished rats. Similarly, treatment with alkaline phosphatase decreased adenylyl cyclase activity but did not eliminate the difference in adenylyl cyclase activity between control and malnourished rats. These data demonstrate that loss of PKA activity is an early response to a low protein diet and that, subsequently, a number of molecular adaptations occur which increase cAMP production. These changes may be adaptive responses to malnutrition that maintain essential cAMP-dependent functions.

Understanding impact of δF508 and G551D CFTR mutations on CFTR/PKA-c interaction.

The CFTR anion channel is an ATP Binding Cassette protein and consists of two pore-forming transmembrane domains, two cytosolic nucleotide binding domains that bind and hydrolyze ATP to drive gating, and a cytosolic regulatory (R) domain. The catalytic subunit of protein kinase A (PKA) binds to, and phosphorylates, the R domain, causing reversible and irreversible channel activation, respectively. CFTR chloride channel mutations cause the lethal and incurable disease cystic fibrosis (CF). The most common CF mutation, ΔF508, is known to impair CFTR protein processing and channel gating, while mutation G551D disrupts ATP-dependent channel gating. In addition, ΔF508 and G551D CFTR were also shown to be defective in PKA-dependent activation. As the stimulating effect of ivacaftor, the only FDA-approved CFTR potentiator drug, depends on CFTR phosphorylation, understanding the reason for the impaired CFTR-PKA interaction in the mutants is of great significance. The reduced rate of phosphorylation of G551D or ΔF508 channels might be a consequence of impaired PKA binding to CFTR. In the presence of ATP the contributions of PKA binding vs. phosphorylation to overall channel activation are difficult to deconvolve. Therefore, we tested reversible activation by PKA of unphosphorylated and phosphorylated mutant channels gating in P-ATP, an ATP analog that supports CFTR gating but cannot be used for phosphotransfer by PKA. Using macroscopic inside-out patch clamp recordings we compared the rates and fractional amplitudes of reversible stimulation by PKA for the mutants and wild-type CFTR. Our data show that channel activation by reversible binding of PKA is preserved in the mutant CFTR channels. Additional experiments are underway to decipher the molecular mechanism for slowed activation of the mutants.

Mislocalization of protein kinase A drives pathology in Cushing's syndrome.

SUMMARYMutations in the catalytic subunit of protein kinase A (PKAc) drive the stress hormone disorder adrenal Cushing’s syndrome. We define mechanisms of action for the PKAc-L205R and W196R variants. Proximity proteomic techniques demonstrate that both Cushing’s mutants are excluded from A kinase-anchoring protein (AKAP)-signaling islands, whereas live-cell photoactivation microscopy reveals that these kinase mutants indiscriminately diffuse throughout the cell. Only cAMP analog drugs that displace native PKAc from AKAPs enhance cortisol release. Rescue experiments that incorporate PKAc mutants into AKAP complexes abolish cortisol overproduction, indicating that kinase anchoring restores normal endocrine function. Analyses of adrenal-specific PKAc-W196R knockin mice and Cushing’s syndrome patient tissue reveal defective signaling mechanisms of the disease. Surprisingly each Cushing’s mutant engages a different mitogenic-signaling pathway, with upregulation of YAP/TAZ by PKAc-L205R and ERK kinase activation by PKAc-W196R. Thus, aberrant spatiotemporal regulation of each Cushing’s variant promotes the transmission of distinct downstream pathogenic signals.

Mutation analysis of circulating tumor DNA and paired ascites and tumor tissues in ovarian cancer

Circulating tumor DNA (ctDNA) is one conventional type of liquid biopsy that can be collected to dynamically monitor disease status. However, its potential clinical value and concordance with ascites samples or tumor biopsy needs to be evaluated further for patients with ovarian cancer. Therefore, the present study compared the mutation profiles among ctDNA, paired tumor tissue and ascites samples to explore their possible clinical value in ovarian cancer. Targeted next-generation sequencing was used to screen for mutations in 18 peripheral blood samples, six paired ascites samples and eight paired tumor tissues collected from patients with ovarian cancer. Functional analyses were performed using public databases. WebGestalt was used to perform Gene Ontology and pathway enrichment analyses. The cBioPortal for Cancer Genomics was used to assess therapeutic targets. Chilibot and Search Tool for the Retrieval of Interacting Genes/Proteins were used to obtain key genes and their functional interactions. Comparative analysis was performed among the three types of samples using Venn diagram. A total of 104 cancer-associated mutant genes in ctDNA samples, 95 genes in tumor tissues and 44 genes in ascites samples were found. A cluster covering 10 genes, namely NOTCH2, NOTCH3, lysine methyltransferase 2A, PTEN, androgen receptor, DNA-activated protein kinase catalytic subunit, hepatocyte nuclear factor 1 homeobox A, SRC, insulin receptor substrate 2 and SRY-box transcription factor 10, was obtained by Chilibot analysis. This gene panel may have the potential to monitor metastasis and identify therapeutic targets in ovarian cancer. Taken together, the present study focused on the mutant genes in ctDNA, ascites and tumor tissues, and suggested that the integrated information of different samples could be examined to comprehensively reflect the mutational landscape in ovarian cancer. However, procedures and protocols to interpret and utilize the integrated information obtained from various forms of liquid biopsies will require optimization prior to their use for future clinical applications.

Abstract 2494: Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer

Abstract Fibrolamellar hepatocellular carcinoma, FLC, is a usually lethal cancer affecting children, and young adults. We have shown that all patients have a disruption in the ecology of protein kinase A activity. Hundreds of patients tested have a fusion of the first exon of DNABJ1, a heat shock protein cofactor, to PRKACA, the catalytic subunit of protein kinase A and expression of this DNAJB1-PRKACA fusion oncokinase is sufficient to produce the tumor. One patient is missing the regulatory subunit of protein kinase A and three patients have a fusion of the first exon of a different protein, ATP1B1, to the catalytic subunit of protein kinase A. We characterized the proteome and phosphoproteome of FLC cells relative to adjacent normal tissue. The changes were sufficiently characteristic to identify cell extracts from FLC, based solely on the proteome or phosphoproteome, and distinguishable from other tumor or normal liver. By calibrating protein level we found that tumor cells have an increase of catalytic subunit to such an extent as to exceed the capacity of protein kinase A regulatory subunits. This has two implications. First, there is free catalytic subunit that is unregulated, creating a high basal level of kinase activity. Second, the catalytic subunit is no longer localized by tethering to the regulatory subunit. Thus, the catalytic subunit has access to novel substrates. As an independent confirmation, we found the free basal kinase activity was higher in FLC cells. As a further test, we showed free catalytic subunit, that is not conjugated to regulatory subunit, is higher in FLC cells. We next tested whether these changes were solely due to overexpression of the catalytic subunit or if there was some additional change in the intrinsic activity of the kinase as a result of the fusion. We purified, without using an affinity tag, either PRKACA (which is myristoylated), DNAJB1-PRKACA, and PRKACA which was not myristoylated (the DNAJB1-PRKACA is not myristoylated). As a further control we also used the PRKACAL206R mutation, which does not engage regulatory subunits and is present in some forms of Cushing’s disease. We purified cytosol from human liver, blocked activity of all the endogenous kinases, and added our purified kinase. Mass spectrometry was then used to identify all newly phosphorylated proteins. While many proteins were equally phosphorylated by all four kinases (PRKACA, DNAJB1-PRKACA, PRKACA not myristoylated, PRKACAL206R) there were some distinct differences. From an analysis of these we found alterations in the optimal recognition sequence for phosphorylation for each variant of the kinase. Significantly, when we examined the human tissue data, we found many proteins that were phosphorylated by the DNAJB1-PRKACA in vitro were also phosphorylated in patient tumor tissue, but not in adjacent non-transformed tissue. This validated these observations of altered phosphorylation from the in vitro assays. Citation Format: Solomon Levin, Mahsa Shironi, Melissa Jarmel, Michael Tomasini, Bassem Shebl, Tova Finkelstein, David Requena, Soeren Heissel, Henrik Molina, Philip Coffino, Barbara Lyons, Sanford M. Simon. Pathogenesis of fibrolamellar: Proteome and phosphome of an oncokinase driven cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2494.

Abstract 3888: Targeting Bcl-xL in fibrolamellar hepatocellular carcinoma

Abstract Fibrolamellar hepatocellular carcinoma (FLC) is a lethal pediatric cancer affecting adolescents and young adults. Besides surgical resection, there is no approved and effective therapy. FLC is driven by a fusion oncokinase, DNAJB1-PRKACA, that arises from a deletion fusing exon 1 of DNAJB1 and exons 2-10 of PRKACA, the catalytic subunit of protein kinase A. Expression of this chimeric oncokinase is capable of recapitulating the key histologic and transcriptomic features of FLC. Using PDXs, we tested therapeutics that have been used clinically for FLC or that target pathways identified via the transcriptomic signature of FLC, in addition to an agnostic library of clinical-stage drugs. Several compounds were identified with a varying degree of efficacy in different PDX lines as compared to our control line, primary human hepatocytes. One of the top hits was irinotecan, and its active metabolite SN38. Irinotecan is a topoisomerase I inhibitor and is clinically utilized in a wide variety of adult and pediatric cancers. A second top hit was Navitoclax, an inhibitor of the anti-apoptotic proteins Bcl-2 and Bcl-xL. Tool compounds that targeted Bcl-xL, but not bcl-2 were efficacious. This suggested a possible link between Bcl-xL, which is overexpressed in some PDX lines, and the resistance profile they showed in our screen. Unfortunately, Navitoclax has an on-target and dose-limiting toxicity of thrombocytopenia, which limits its clinical application. DT2216 is a VHL-based PROTAC that degrades Bcl-xL. DT2216 has two potential benefits over Navitoclax: 1) DT2216 is a more potent than Navitoclax against various Bcl-xL dependent tumor cells; and 2) it is less toxic to platelets than Navitoclax by targeting Bcl-xl to VHL that is poorly expressed in platelets to avoid induction of severe thrombocytopenia. A phase 1 clinical trial of DT2216 in relapsed/refractory malignancies is now ongoing (NCT04886622). We tested if the combination of DT2216 and irinotecan might have a therapeutic potential in FLC. Indeed, in vitro screening of the proposed combo, DT2216 and SN38, showed an additive or a synergistical effect in 4 validated FLC cell lines. We tested both the efficacy of DT2216 on Bcl-xL levels in FLC as well as platelet counts to identify any developing thrombocytopenia. DT2216 was less disruptive to platelets than Navitoclax at therapeutically relevant pre-clinical doses. Next, we tested 4 of our PDX models for FLC using various dosing regimens to identify the most efficacious regimen with the minimum side effects. Mice cohorts were evaluated for weight loss and tumor volume. Mice treated with DT2216 and SN38 showed consistent therapeutic benefit which was not evident in controls and less notable as monotherapy. In conclusion, TOPO1 and Bcl-xL prove to be promising targets of interest in FLC. Targeting both with DT2216 and irinotecan leads to tumor shrinkage in pre-clinical models and offer a potential therapeutic/pharmacological intervention for FLC. Citation Format: Bassem Shebl, Gadi Lalazar, Denise Ng, Tova Finkelstein, Guangrong Zheng, Peiyi Zhang, Carly Rosemore, Michael Ortiz, Daohong Zhou, Sanford Simon, Philip Coffino. Targeting Bcl-xL in fibrolamellar hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3888.

Meprin β Regulates Protein Kinase A Mediated TGF‐β Signaling in Ischemia Reperfusion‐Induced Kidney Injury

Ischemia/reperfusion (IR) is a leading cause of acute kidney injury (AKI) and is associated with high morbidity and mortality rates. The underlying mechanisms involved in IR‐induced AKI are not fully understood. Meprins, zinc metalloproteases that are abundantly expressed in the brush border membranes of proximal kidney tubules, have been implicated in the pathology of IR. Meprins are capable to proteolytically processing the catalytic subunit of protein kinase A (PKA‐C) and several mediators of inflammation. The protein kinase A (PKA) signaling pathway plays an important role in renal fibrosis, promoting the production of transforming growth factor‐β (TGF‐β) and/or TGF‐β‐dependent molecules. The TGF‐β signaling is involved in wound healing and tissue repair of injured kidneys. Activation of Smad2 and Smad3 is the major downstream event of the TGF‐β signaling pathway. The objective of the current study was to determine how meprin β expression impacts PKA C‐mediated TGF‐β signaling in IR‐induced kidney injury. To this end, we used surgical procedures for to achieve unilateral IR with contralateral nephrectomy in wild‐type (WT) and meprin β knockout (βKO) mice. Meprin βKO mice are deficient in the meprin B (β‐β) and the heterodimeric isoform of meprin A (α‐β). The mice were sacrificed at 96 h post‐IR and kidney tissues obtained for analysis. Real‐time PCR, and immunohistochemical staining, were used to evaluate the expression levels of PKA C and TGF‐β. Statistical analysis utilized 2‐way ANOVA. Real‐time PCR data showed significant increases in mRNA levels of PKA C only in βKO kidneys after IR (p< 0.05). In contrast, the mRNA levels for TGF‐β were higher in both WT and βKO at 96 h post‐IR (p< 0.01). Interestingly, the baseline mRNA levels for TGF‐β were higher in meprin βKO mice (p< 0.001). Immunohistochemical staining for PKA C and TGF‐β was quantified using Image J software. The data showed significant increases in the levels of PKA C proteins in select kidney tubules of both genotypes (p≤ 0.001) after IR. Similarly, staining intensity for TGF‐β were higher in select kidney tubules of both WT (p=0.01) and meprin β deficient mice at 96 h IR (p=0.001). Immunofluorescence counterstaining of kidney tissues with anti‐meprin β, anti‐PKA C and anti‐TGF‐β antibodies showed a positive correlation between meprin β expression and tubular PKA C and TGF‐β levels in kidneys subjected to IR. These findings suggest that meprin β regulates PKA C‐mediated TGF‐β signaling pathway in IR‐induced kidney injury and provides new insights on the mechanisms underlying meprin β modulation of the pathophysiology of IR‐induced renal injury.

Non‐canonical Recruitment of PKA Catalytic Subunits to RIα‐driven Biomolecular Condensates

A regulatory subunit of cAMP‐dependent protein kinase (PKA), RIα, was recently shown to undergo liquid‐liquid phase separation (LLPS), which is critical for cAMP compartmentation. These RIα biomolecular condensates form upon cAMP production, contain high levels of cAMP and active PKA catalytic (PKAcat) subunits, buffer increases in cAMP concentration, and suppress tumorigenic phenotypes. Through mutagenesis studies of key residues within different domains of RIα, we found that cAMP binding to both CNB‐A and B domains is required to stimulate RIα LLPS and that the dimerization via both the dimerization/docking (D/D) domain and the N3A motif of the CNB‐A is required for RIα LLPS. Our data suggests that the inhibitory sequence (IS), which binds and inhibits the catalytic subunit, is also involved in the regulation of RIα LLPS as well as recruitment of the catalytic subunit to the RIα puncta, likely through a non‐canonical interaction. RIα mutants carrying point mutations (G98S and A99S) in the IS showed enhanced RIα LLPS but weakened R:C complex and decreased capability to recruit PKAcat to RIα puncta. Intriguingly, these mutants generated puncta that did not disassemble when cAMP levels decreased, whereas puncta from wildtype RIα showed complete disassembly, suggesting that the IS motif impacts the dynamics of RIα LLPS. Our study led to a further understanding of the stimulation, formation, PKAcat recruitment, and disassembly of PKA biomolecular condensates, which may lead to novel therapeutic approaches for diseases driven by a loss of PKA RIα LLPS, such as fibrolamellar carcinoma.

Impact of Redox Modification on PKA Substrate Selection

Protein kinases regulate multiple signaling pathways involved in health and disease. For instance, cyclic adenosine monophosphate‐dependent protein kinase (PKA) controls a diverse set of cellular functions, including gene expression, cell cycle progression, metabolism, and apoptosis. Dysregulation of PKA has been linked to many diseases, including diabetes, cancer, and neurodegenerative disorders. PKA is a tetramer consisting of two regulatory and two catalytic subunits. Cyclic AMP binding to the regulatory subunits leads to dissociation of the catalytic subunits, unleashing them to phosphorylate serine and threonine residues on their target substrates. Previous studies suggest that the alpha isoform of the PKA catalytic subunit (PKA Cα) is oxidized on C199 within the P+1 substrate binding region. We hypothesized that, by modifying the substrate selectivity of PKA Cα, oxidation may regulate which pathway is activated in response to a given signal. Specifically, we investigated the impact of redox modification on the substrate selection of PKA Cα. To this end, we used fluorescence polarization (FP) spectroscopy to assess the ability of oxidized and reduced forms of PKA Cα to bind a variety of model peptide ligands (e.g., PKI, Kemptide, and CREBtide). The binding of each ligand was monitored under equilibrium binding conditions and the apparent equilibrium binding parameters (i.e., KD,app) were determined for each kinase‐ligand pair. These studies suggest that H2O2‐dependent oxidation of PKA Cα alters its interactions with some ligands while having little effect on others. To gain further insights into the factors driving the differential changes in the interaction between PKA Cα and its ligands, we are currently using surface plasmon resonance (SPR) to measure changes in the apparent rate constants, kon,app and koff,app, as well as KD,app. Together, these studies offer important insights into the mechanisms of crosstalk between redox and phosphorylation‐dependent signaling at the level of kinase substrate selection and have implications for kinase regulation during normal and pathological states. Interestingly, the site of oxidation is highly conserved among AGC kinase family members. Therefore, in the future, we will use a multipronged approach to explore the impact of redox modification on the substrate selection of other AGC family members, such as PKA Cβ and AKT1, in neuronal signaling and the etiology of various diseases.

My Journey with cAMP‐dependent Protein Kinase

I was first introduced to cAMP‐dependent protein kinase (PKA) when I was just a young scientist at the beginning of my career, and it has remained as a lifelong companion. PKA has taken me from protein chemistry to atomic structures to brain imaging, and the quest for new knowledge is still ongoing. PKA was the second kinase to be discovered, and we solved the first protein kinase structure in 1991. In the subsequent decades we have come to appreciate the highly dynamic features that drive this molecular allosteric switch. In cells the activity of the PKA catalytic subunit is typically packaged as a holoenzyme complex with functionally non‐redundant cAMP‐binding regulatory (R) subunits, and the holoenzymes are localized to specific sites in the cell in close proximity to dedicated substrates. Many dedicated “cAMP signaling islands” exist within one cell, some at the plasma membrane but others at organelles such as the mitochondria and Golgi and in the nucleus, and each is tightly regulated. Nowhere is PKA signaling more complex than in the brain. PKA is ubiquitous in every mammalian cell, and in so many ways has served as the “poster child” for the kinome that contains over 500 proteins. How then can we explain why the Cb subunit lies near the bottom of the Dark Kinome where important protein kinase are buried but not yet studied? The Cb family contains several isoforms that all differ in only their first exon and includes many splice variants. While Cb1 is expressed ubiquitously in most cells at low levels, Cb2 is expressed in lymphoid tissues while the Cb3/4 isoforms are expressed almost exclusively in brain. Indeed ~50% of PKA signaling in the brain is mediated by Cb isoforms. Using the retina with its well‐organized and terminally differentiated neurons as a “window into the brain” we find that Cb isoforms are highly expressed and that Cb is localized differently from Ca. In addition, we find that Cb is localized to mitochondria in contrast to Ca, RIIa, and RIIb, which are all excluded from mitochondria. Four distinct mutations in Cb lead to a complex phenotype that includes cardiac and skeletal defects as well as a polydactyl presentation. These mutations also inhibit Sonic hedgehog (Shh) signaling adding further support to the importance of Cb in neuronal signaling and development. We are our interdisciplinary strategy to explore this new frontier for PKA signaling in the brain.

SESN1 attenuates the Ox‑LDL‑induced inflammation, apoptosis and endothelial‑mesenchymal transition of human umbilical vein endothelial cells by regulating AMPK/SIRT1/LOX1 signaling.

Endothelial cells are an important component of the heart and vasculature and form a crucial link between the cardiovascular system and the immune system. Sestrin 1 (SESN1) has an important role in atherosclerosis by inhibiting NOD-like receptor family pyrin domain containing 3 inflammasome activation. However, whether SESN1 is involved in human umbilical vein endothelial cell (HUVEC) injury caused by atherosclerosis has remained to be elucidated. The present study aimed to investigate the functions of SESN1 in the inflammatory response, apoptosis and endothelial-mesenchymal transition (EndMT) of HUVECs following stimulation with oxidized low-density lipoprotein (Ox-LDL). SESN1 expression at the mRNA and protein levels was detected using reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. Following SESN1 overexpression in Ox-LDL-stimulated HUVECs, cell viability was determined using a Cell Counting Kit-8 assay. Terminal deoxynucleotidyl transferase-mediated nick-end labeling staining was employed to detect cell apoptosis and western blot analysis was used to determine the levels of apoptosis-related proteins. RT-qPCR, ELISA and western blot were utilized to determine the levels of inflammatory factors. Immunofluorescence staining, RT-qPCR and western blot analysis were employed to assess the EndMT of Ox-LDL-stimulated HUVECs. The results revealed that SESN1 exhibited a low expression in HUVECs following Ox-LDL stimulation. SESN1 overexpression suppressed inflammation, apoptosis and EndMT in Ox-LDL-induced HUVECs. In addition, SESN1 stimulated adenosine monophosphate-activated protein kinase catalytic subunit α1/sirtuin 1 signaling to suppress Ox-LDL receptor-1 expression. An AMPK and SIRT1 inhibitor reversed the effects of SESN1 overexpression on the inflammatory response, apoptosis and EndMT of HUVECs exposed to Ox-LDL. Taken together, the present study demonstrated that SENS1 exerts a suppressive effect on Ox-LDL-induced inflammation, apoptosis and EndMT of HUVECs, suggesting that SENS1 may be used as a novel biomarker for endothelial injury-related disorders.