PI3K Signaling Pathway Research Articles

Abstract B027: Bioluminescence resonance energy transfer (BRET) as a tool for assessing mutant KRAS-effector affinity and drug efficacy

Abstract KRAS mutations occur in roughly 15% of cancer patients, with KRAS G12D, KRAS G12V, and KRAS G12C substitutions constituting the majority of these cases. Cancer types also display puzzling preferences for certain RAS mutations; for instance, 49% of pancreatic ductal carcinomas (PDAC) are driven by KRAS G12D substitutions compared to 35% of colon adenocarcinomas (COAD) and 17% of lung adenocarcinomas (LUAD)1. In contrast, KRAS G12C mutations drive only 3% of PDAC and 10% of COAD, but 41% of LUAD cases1. Some of this discrepancy can be explained by mutagen exposure, as there is evidence that tobacco smoke carcinogens drive the predominance of KRAS G12C mutations in lung cancer. However, it is possible that inherent functional differences between the mutant proteins also contribute to such preferences across cancer types. These oncogenic substitutions do endow subtle and unique differences in biochemical properties, but it is unknown whether this translates to differences in cellular signaling potential. KRAS activates the MAPK and PI3K signaling pathways by binding directly to RAF1 or the p110 catalytic subunit of PI3K. These interactions are important signaling nodes that result in cellular proliferation, survival, movement, differentiation, and immune evasion, and therefore drive cancer when constitutively activated by mutant KRAS. Biochemical assessments of relative effector affinity between KRAS mutants lack crucial elements present in a cellular environment, such as GDP/GTP ratios, membrane interactions, subcellular localization, and engagement with other proteins. Here, we optimize a cell-based Bioluminescent Resonance Energy Transfer (BRET) system in HEK293T cells using NanoLuc- and mVenus-tagged proteins to quantify relative effector affinities between KRAS mutants. We assess the affinities of KRAS4b G12C, KRAS4b G12D, and KRAS4b WT for effectors p110α and RAF1(52-188), along with RAS binding deficient control mutants p110α T208D/K227A and RAF1(52-188)R89L, and show statistical differences in effector engagement between the wildtype and mutant proteins. We also demonstrate the efficacy of this optimized system as a drug screening tool by assessing compounds in clinical development for their ability to disrupt mutant KRAS-effector interactions within a cellular environment. 1Prior IA, Hood FE, Hartley JL. The frequency of Ras mutations in cancer. Cancer Research 2020. Citation Format: Megan Rigby, John Columbus, Vanessa Wall, Dominic Esposito, Thomas Turbyville, Dhirendra Simanshu, Dwight Nissley, Frank McCormick, Anna Maciag. Bioluminescence resonance energy transfer (BRET) as a tool for assessing mutant KRAS-effector affinity and drug efficacy [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B027.

Role of Src-PI3K signaling activation in regulation of STAT5b-deficient NK cell function in response to IL-2

BackgroundMutations in the Signal Transducer and Activator of Transcription (STAT)5b, are associated with low numbers of NK cells and poor cytolytic function. IL-2 enhances cytotoxicity and requires STAT5b as a downstream effector. It can also induce Src protein activation to promote lytic granule convergence, an essential step in mobilizing lytic granules to the immunologic synapse for cytotoxicity. Subsequently, after lytic granule convergence and conjugation of the NK cell to the target cell activation of PI3K signaling pathway activation leads to target lysis. We have previously shown that IL-2 can partially rescue NK cell cytotoxicity in STAT5b-deficient cells. However, how IL-2 stimulation induces PI3K activity outside of IL-2/STAT5b signaling is not entirely clear. Using STAT5b-deficient NK cells (p. Q41X) and STAT5b-KD NK cell lines as a human model,we elucidate the role of the non-canonical IL-2 signaling pathway through Src, MEK, ERK, and pS6 activation. MethodsWe used multiparametric flow cytometry, functional NK cell assays (standard Cr51 release assay), and microscopy to elucidate the effect of non-canonical IL-2 signaling in human STAT5-deficient NK cells (p.Q41X) and conditional doxycycline STAT5-KD YTS and NK92 NK cell lines. STAT5b expression was evaluated by Western blot. Src, MEK, ERK1/2, and pS6 activationwere evaluated in primary NK cells and STAT5-KD NK cell lines by WB and FC in presence or absence of IL-2. ResultsThe STAT5b-KD NK cell lines mimics the abnormalities observed in primary STAT5b-deficient NK cells (p.Q41X). Mutant primary and cell lines showed abnormal NK cell maturation, decreased convergence of lytic granules to the microtubule-organizing center (MTOC) leading to impaired NK cell cytotoxic capacity partially restored after IL-2 stimulation. We measure downstream mediators of PI3 K activation (MEK, ERK, and pS6) after IL-2 and observed increase levels of pAKT and pERK compared to HD andWT with lower levels of pS6. The activation of these protein correlated with lytic granule convergence and restored cytotoxicity suggesting that IL-2 through Src activates PI3 K by way of pAKT and pERK. ConclusionsOur results suggest restored granule convergence and partial improvement in NK cell killing happens through the non-canonical IL-2 pathway by Src-PI3K-MEK-ERK activation.

ELMO1 Deficiency Reduces Neutrophil Chemotaxis in Murine Peritonitis.

Peritoneal inflammation remains a major cause of treatment failure in patients with kidney failure who receive peritoneal dialysis. Peritoneal inflammation is characterized by an increase in neutrophil infiltration. However, the molecular mechanisms that control neutrophil recruitment in peritonitis are not fully understood. ELMO and DOCK proteins form complexes which function as guanine nucleotide exchange factors to activate the small GTPase Rac to regulate F-actin dynamics during chemotaxis. In the current study, we found that deletion of the Elmo1 gene causes defects in chemotaxis and the adhesion of neutrophils. ELMO1 plays a role in the fMLP-induced activation of Rac1 in parallel with the PI3K and mTORC2 signaling pathways. Importantly, we also reveal that peritoneal inflammation is alleviated in Elmo1 knockout mice in the mouse model of thioglycollate-induced peritonitis. Our results suggest that ELMO1 functions as an evolutionarily conserved regulator for the activation of Rac to control the chemotaxis of neutrophils both in vitro and in vivo. Our results suggest that the targeted inhibition of ELMO1 may pave the way for the design of novel anti-inflammatory therapies for peritonitis.

Design, Synthesis and Biological Evaluation of Multi-Target Anti-Cancer Agent PYR26.

This study investigates the synthesis of a new compound, PYR26, and the multi-target mechanism of PYR26 inhibiting the proliferation of HepG2 human hepatocellular carcinoma cells. PYR26 significantly inhibits the growth of HepG2 cells (p < 0.0001) and this inhibition has a concentration effect. There was no significant change in ROS release from HepG2 cells after PYR26 treatment. The mRNA expressions of CDK4, c-Met and Bak genes in HepG2 cells were significantly inhibited (p < 0.05), while mRNA expression of pro-apoptotic factors such as caspase-3 and Cyt c was significantly increased (p < 0.01). The expression of PI3K, CDK4 and pERK proteins decreased. The expression level of caspase-3 protein was increased. PI3K is a kind of intracellular phosphatidylinositol kinase. PI3K signaling pathway is involved in signal transduction of a variety of growth factors, cytokines and extracellular matrix and plays an important role in preventing cell apoptosis, promoting cell survival and influencing cell glucose metabolism. CDK4 is a catalytic subunit of the protein kinase complex and is important for G1 phase progression of the cell cycle. PERK refers to phosphorylated activated ERK, which is translocated from cytoplasm to the nucleus after activation, and then participates in various biological reactions such as cell proliferation and differentiation, cell morphology maintenance, cytoskeleton construction, cell apoptosis and cell canceration. Compared with the model group and the positive control group, the tumor volume of the nude mice in the low-concentration PYR26 group, the medium-concentration group and the high-concentration group was smaller, and the organ volume was smaller than that in the model group and the positive control group. The tumor inhibition rates of low-concentration group PYR26, medium-concentration group and high-concentration group reached 50.46%, 80.66% and 74.59%, respectively. The results showed that PYR26 inhibited the proliferation of HepG2 cells and induced apoptosis of HepG2 cells by down-regulating c-Met, CDK4 and Bak, up-regulating the mRNA expression of caspase-3 and Cyt c genes, down-regulating PI3K, pERK and CDK4 proteins and up-regulating the protein level of caspase-3. In a certain range, with the increase in PYR26 concentration, the tumor growth was slower and the tumor volume was smaller. Preliminary results showed that PYR26 also had an inhibitory effect on the tumors of Hepa1-6 tumor-bearing mice. These results suggest that PYR26 has an inhibitory effect on the growth of liver cancer cells, therefore it has potential to be developed into a new anti-liver cancer drug.

Xiaoyaosan ameliorates depressive-like behavior and susceptibility to glucose intolerance in rat: involvement of LepR-STAT3/PI3K pathway in hypothalamic arcuate nucleus

BackgroundAccumulating evidence has demonstrated that arcuate nucleus (ARC) of the hypothalamus is likely responsible for the close association between chronic stress, depression, and diabetes. Xiaoyaosan (XYS), a Chinese herbal formula, remarkably improves depressive-like behavior and glucose intolerance, but the mechanism remains unclear. Leptin receptor (LepR) regulates energy expenditure and depression by mediating the action of leptin on the ARC. Therefore, we hypothesized that XYS may regulate depressive-like behavior and glucose intolerance via the leptin and its cascade LepR-STAT3/PI3K pathway in the ARC.MethodsA rat model of depressive-like behavior and susceptibility to glucose intolerance was induced by exposure to chronic unpredictable mild stress (CUMS) for six weeks. XYS (2.224 g/kg) was orally gavaged for six weeks, and fluoxetine (2.0 mg/kg) was administrated to the positive control group. Depressive-like behaviors were assessed using the open field test (OFT), sucrose preference test (SPT) and forced swim test (FST). Fasting blood glucose (FBG) and oral glucose tolerance test (OGTT) were performed to evaluate the effects of XYS on blood glucose. Peripheral leptin and blood lipids were detected using enzyme-linked immunosorbent assay and an automatic biochemical analyzer, respectively. The effects of XYS on the LepR-STAT3/PI3K pathway were detected by quantitative real-time PCR and western blotting.ResultsXYS ameliorated CUMS-induced depressive-like behaviors and elevated blood glucose. XYS improved the food intake but have no significant effects on the body weight. Peripheral leptin and its central receptor were also suppressed by XYS, accompanied by the downregulation of JAK2/STAT3 and PI3K/AKT pathway in the ARC. Additionally, XYS increased AGRP and NPY expression but inhibited POMC in the ARC.ConclusionsXYS improves depressive-like behaviors and susceptibility to glucose intolerance induced by CUMS, which may be achieved by the downregulation of the LepR-STAT3/PI3K signaling pathway in the ARC.

Abstract 2684: Synergistic efficacy of HSP90 and PI3K inhibitors in adrenocortical carcinoma

Abstract Adrenocortical cancer (ACC) is a rare and aggressive cancer (0.5-2 cases/million/year) with poor 5-year survival. Most patients inevitably succumb to widespread metastasis due to a lack of effective systemic treatment; therefore, it is critically important to identify new therapies for clinical trials. In this direction, we identified a potent synergistic combination of Heat Shock Protein 90 (HSP90) and PI3K inhibitors via quantitative high-throughput drug screening (qHTS) for ACC, using a pharmaceutical library of ~5000 drugs (FDA approved or investigational), and evaluating the drug synergy by computerized drug combination matrix analysis. Preclinical in vitro studies were performed to validate the efficacy of HSP90 inhibitors (STA9090, AUY922, HSP990), with PI3K inhibitors; PIK75 (investigational) or clinically available BGT226, and their pairwise combinations, in NCI-H295R and SW13 ACC cells. The drug combination of STA9090 and PIK75 significantly inhibited cell proliferation (monolayer and 3-dimensional culture and induced apoptosis more effectively than the individual drugs, with simultaneous upregulation of apoptotic protein markers (cleaved-Caspase3, cleaved-PARP), inhibition of the phosphorylated members of PI3K signaling pathway (AKT1, mTOR, S6K, 4EB1 and GSK3α/β), and the G2/M phase cell cycle arrest in ACC cells. Additionally, the synergetic effect of the drug combination was effective in reducing the metastatic potential as revealed by invasion/migration assay, which was confirmed by the downregulation of the epithelial-to-mesenchymal transition proteins (Vimentin, N-cadherin, SNAIL and Zeb1). An inhibition of cortisol production and altered oncogenic proteomic profiling was also observed. We validated the synergy of BGT226 with STA9090 and observed similar phenotypic changes by the drug combination in ACC cells. Interestingly, RNA sequencing and pathway analysis further revealed distinct gene expression profiles of STA9090 combined with PIK75 or BGT226, indicating different mechanism of cell death in both the PI3K drug combination treatment groups. Consistent with our in vitro findings, observations from the gene expression data from TCGA revealed significant mRNA overexpression of genes in HSP90 and PI3K signaling pathways (HSP90AA1, HSP90AB1 HSP90B1, TRAP1, PI3KCA, PI3KCB, AKT2, CDK1, CDK4, NR3C1) in 64% (n=59/92) ACC samples, with a positive correlation of mesenchymal markers TWIST1, ZEB1, VIM with HSP90AB1 and PI3KCA, suggesting our drug combination can be applicable in patients with ACC. A similar pattern of gene expressions was confirmed in independent GEO datasets. Conclusively, combination of HSP90 and PI3K inhibitors demonstrated a promising in vitro synergistic efficacy by inhibiting the key oncogenic targets of ACC. Further validation of in vivo efficacy is warranted. Citation Format: Prachi Mishra, Dipranjan Laha, Brieann Sobieski, Min Shen, Ya-Qin Zhang, Matthew Hall, Naris Nilubol. Synergistic efficacy of HSP90 and PI3K inhibitors in adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2684.

Abstract 8: Differential flt3 mutational status in acute myeloid leukemia predicts sensitivity to flt3 inhibitor gilteritinib in vitro and in vivo

Abstract Acute Myeloid Leukemia (AML) is a heterogenous hematopoietic cancer characterized by abnormal differentiation and uncontrolled proliferation of hematopoietic progenitor cells. Recent approaches to treatment, which aim to improve outcomes in refractory AML, include strategies to molecularly target specific receptor-mediated signaling pathways which regulate cell growth and proliferation. One target-specific approach is the inhibition of fms-like tyrosine kinase 3 (FLT3). FLT3 is expressed on the surface of hematopoietic progenitor cells and plays an important role in regulating proliferation, survival, and differentiation of multipotent stem cells. The presence of internal tandem duplications (ITDs) at the juxta membrane domain within the FLT3 receptor are common driver mutations in 30% of AML patients. In this study, we utilized 3 luciferase-expressing human AML models, MV4-11, MOLM-13, and, OCI-AML3 to determine the prognostic impact of FLT3 inhibitor Gilteritinib on the allelic ratio of ITD versus wildtype allele. OCI-AML3 expresses wild-type FLT3 whereas MOLM-13 is heterozygous for FLT3-ITD mutations, and MV4-11 possesses a homozygous in-frame tandem repeat insertion in the FLT3 allele. Here we demonstrate that FLT3-ITD mutational status impacts the efficacy of Gilteritinib in both in vitro and in vivo AML models. The in vitro assays showed that treatment with Gilteritinib resulted in significant dose dependent reduction in overall cell viability and bioluminescence in the MV4-11 cell line, while MOLM-13 was only moderately sensitive, and OCI-AML3 was least sensitive to Gilteritinib treatment. Additionally, we evaluated the downstream MAPK and PI3K signaling pathways as FLT3-ITD oncogenic mutations result in ligand-independent constitutive activation of signaling pathways mediating leukemic transformation. For in vivo validation, MV4-11, MOLM-13, and OCI-AML3 luciferase expressing cells were implanted systemically into NCG mice and once tumors were established, we evaluated the survival benefit in response to Gilteritinib and various standard of care agents. In accordance with our in vitro findings, the quantification of tumor progression by bioluminescent imaging showed the highest therapeutic activity of Gilteritinib in MV4-11 tumor bearing mice through selective targeting of FLT3-ITD mutations. Taken together, our in vitro and in vivo results demonstrate that differential FLT3-ITD mutational status in AML models impacts Gilteritinib treatment response and downstream signaling events with the highest activity in MV4-11 followed by MOLM-13, and lack of activity in OCI-AML3 harboring wild-type FLT3. Citation Format: Bincy John, Pasupathi Sundaramoorthy, Elizabeth Rainbolt, Andrew Wong, Zachary Ward, Justin Avery, Savannah Todd, Shasta Kidder, Diana Gietl, Chassidy Hall, David Harris. Differential flt3 mutational status in acute myeloid leukemia predicts sensitivity to flt3 inhibitor gilteritinib in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 8.

Abstract 2209: A proteomic-based prognostic signature of pancreatic adenocarcinoma

Abstract Introduction: Pancreatic cancer has a very poor prognosis, with no established prognostic biomarkers in clinical use. This project aims to identify a prognostic proteomic-based signature for pancreatic adenocarcinomas. Methods: Fresh frozen tumors and matched normal samples from 125 patients were prepared for proteomic analyses using data-independent acquisition mass spectrometry (DIA-MS). Differential expression analyses were conducted on the normalized protein matrix to identify the top differentially expressed proteins (DEP) within the tumor samples. DEP were subjected to crosstalk and pathway enrichment analysis (PEA). Survival analysis based on initial univariate and subsequent 100 runs of multivariate Cox regression with Least Absolute Shrinkage and Selection Operator (LASSO) was performed to obtain a reduced list of candidate proteins associated with Overall Survival (OS). The proteins that appeared in greater than 95% of the LASSO runs were then used in a multivariate Cox model with recursive feature selection, which yielded the final 29 proteins. A risk score was built from the final 29 proteins. Consensus clustering was performed on the median absolute deviation-based top 20% highly variable proteins in tumor samples to identify proteomic-based subtypes. Results: Proteomic analyses revealed 5614 proteins identified from 599 sample runs. Differential expression analyses revealed 398 DEP in tumor samples (FDR-adjusted p-value &amp;lt;0.05, and |logFC|&amp;gt;1). PEA showed that these proteins were related to focal adhesion, extracellular matrix interaction (ECM), angiogenesis, and PI3K signaling pathways. A total of 803 proteins were significantly associated with OS in a univariate Cox regression analysis (p&amp;lt;0.05). PEA on the top 200 proteins associated with poorer OS revealed pathways related to focal adhesion, PI3K signaling, ECM and hypoxia-induced factor-1. Using LASSO multivariate Cox regression modeling, a 29-protein signature was identified, from which a risk score was calculated that dichotomized patients into high- and low-risk groups in terms of OS (Hazard ratio (HR) 2.8, 95% Confidence Interval (CI) [2.3, 3.3], concordance index of 0.91). This risk score was also prognostic for recurrence and three-year survival (both p&amp;lt;0.0001). A multivariate Cox regression model adjusted for other clinical variables revealed a significant association of the risk score with OS (HR 2.91, 95% CI [2.4, 3.5], p&amp;lt;0.001) while maintaining the concordance index (0.907). Consensus clustering analyses revealed four proteomic-based clusters, with cluster 3 showing the worst OS (p&amp;lt;0.001), independent of other clinical variables. PEA on the DEP within cluster 3 showed upregulation of proteins related to cell adhesion, angiogenesis, and immune-related pathways. Conclusion: A 29-protein signature identified a sub-group of patients with pancreatic adenocarcinoma with a poorer prognosis independent of clinical variables. Citation Format: Adel T. Aref, AKM Azad, Asim Anees, Mohashin Pathan, Jason Grealey, Daniela-Lee Smith, Erin M. Humphries, Daniel Bucio-Noble, Jennifer M. Koh, Erin Sykes, Steven G. Williams, Ruth Lyons, Natasha Lucas, Dylan Xavier, Sumit Sahni, Anubhav Mittal, Jaswinder S. Samra, John V. Pearson, Nicola Waddell, Peter G. Hains, Phil J. Robinson, Qing Zhong, Roger R. Reddel, Anthony J. Gill. A proteomic-based prognostic signature of pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2209.

Abstract 1079: Combination of tipifarnib with KRAS G12C inhibitors to prevent adaptive resistance

Abstract Selective KRAS G12C inhibitors, including AMG510 (sotorasib) and MRTX849 (adagrasib), have exhibited clinical activity in patients with non-small cell lung cancer (NSCLC); however, drug resistance and relapse inevitably occur through various mechanisms, which limits the ability of these therapeutic agents to achieve durable responses. Resistance mechanisms include feedback reactivation of compensatory, oncogenic signaling pathways and the attainment of a drug-tolerant state, each of which allows cancer cells to survive the anti-tumor effects of these highly active drugs. Compensatory mechanisms ameliorating KRAS blockade include HRAS/NRAS and the PI3K-AKT-mTOR pathway. Tipifarnib is a clinical stage farnesyltransferase inhibitor known to inhibit multiple farnesylation-dependent proteins in tumor cells. We have recently shown that tipifarnib can prevent feedback-mediated adaptive resistance to the PI3Kα inhibitor, alpelisib, through inhibition of both HRAS and RHEB, allowing simultaneous blockade of two key nodes of the oncogenic MAPK- and PI3K-signaling pathways. In another example, tipifarnib has been demonstrated by others to inhibit the ability of tumor cells to enter a drug-tolerant state induced by the EGFR inhibitor, osimertinib, in EGFR-mutant NSCLC models. In these models, inhibition of farnesylation of certain RHO proteins appear to contribute to the death of drug-tolerant cells prior to the emergence of acquired resistance mutations. Building on these two mechanisms of how tipifarnib may prevent adaptive resistance, we propose that tipifarnib can be used as an effective partner drug to delay or prevent the onset of adaptive resistance to KRAS G12C inhibitors. We have conducted in vitro 2D and 3D viability and regrowth experiments using combinations of tipifarnib with KRAS G12C inhibitors and have observed synergistic, anti-proliferative effects in KRAS G12C NSCLC cell lines as well as enhanced activity of combination in a KRAS G12C NSCLC PDX model. We are currently expanding the scope of in vitro and in vivo combination studies to further evaluate the molecular mechanism(s) of resistance that tipifarnib targets when combined with KRAS G12C inhibitors. Citation Format: Hetika Vora Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. Combination of tipifarnib with KRAS G12C inhibitors to prevent adaptive resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1079.

Abstract 5308: Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers

Abstract Small Cell Lung Cancer (SCLC) is an aggressive tumor type, usually metastatic at diagnosis, with a median survival of less than a year. SCLC tumors are heterogeneous and are composed of neuroendocrine (A, A2, N) and non-neuroendocrine (Y) subtypes, with the cooperation by heterogeneous cells being key to drive tumor survival. Unfortunately, the current SCLC treatment approach does not take tumor heterogeneity into consideration. Importantly, there are no consensus markers that can identify SCLC subtypes or early tumors. Extracellular vesicles (EVs), small vesicles known to promote cell to cell communication, have been investigated as non-invasive biomarkers for cancer diagnostics, staging, and treatment monitoring. EVs are attractive candidates for biomarker research due to the selective packaging of RNA, DNA, and proteins that are then secreted in the blood. Therefore, our aim was to identify new biomarkers based on EV surface proteins with potential for early detection and tumor characterization. We used differential ultracentrifugation to isolate EVs from four SCLC subtypes A, A2, N, and Y, which were submitted to label-free mass spectrometry (EV-proteomics). We identified a total of 3011 proteins, with the two most distinct subtypes being A2 and N, with 786 differentially expressed EV-proteins. Meanwhile, the 2 most similar subtypes were N and A, with only 75 differentially expressed EV-proteins. Gene set enrichment analysis of EVs derived from the A2 subtype showed an association with pathways in cancer, chemokine signaling, and neuronal systems. In addition, PANTHER analysis identified enrichment in nicotine pharmacodynamic pathways on these EVs, which corroborate the association of A2 cell lines with drug resistance. EVs derived from the N and A subtypes were enriched in spliceosome, RNA processing, and metabolism, while EVs from Y cell lines were enriched in extracellular matrix organization and metabolic pathways, similar to pathways from cell line RNAseq data. 13 proteins were differentially expressed in all group comparisons, including proteins involved in PI3K and nicotinic acetylcholine receptor signaling pathways. Further comparison of EV-proteomics with top genes from CIBERSORT analysis using cell line RNAseq data showed an overlap of 9 surface proteins, with 3 possible SCLC pan markers. In conclusion, EV-proteins isolated from SCLC cell lines presented a unique signature for each subtype, especially EVs derived from A2 subtype, which has a signature associated with drug resistance. We identified surface proteins on SCLC EVs with potential as SCLC pan and tumor characterization, which will be validated on EVs derived from cell lines and patient plasma by Western blotting and single-EV microflow cytometry. Citation Format: Patricia M. Ozawa, Sarah M. Groves, Vito Quaranta, Alissa M. Weaver. Analysis of extracellular vesicle surface markers as small cell lung cancer biomarkers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5308.

Abstract 1669: In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor

Abstract Tumor cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Unchecked tumor cell proliferation, as well as macrophage proliferation, contributes to tumor growth and spread. Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth until resistance due to upregulation of the alternative pathway develops. We identified a novel allosteric cKIT/FLT3/CSF1R inhibitor that induces synthetic lethality in AML, lung carcinoma, pancreatic carcinoma and other tumor cells and in proliferating macrophages in vitro and in tumors in vivo by simultaneously inhibiting the MEK-ERK and PI3K-AKT pathways and activating stress activated kinase-mediated cell cycle arrest. A series of related cKIT/FLT3/CSF1R/PDGFR allosteric inhibitors suppressed receptor tyrosine phosphorylation as well as downstream MEK-ERK and PI3K-AKT signaling and mTorc1 and cMyc gene expression pathways that control cell growth. Select cKIT/FLT3/CSF1R/PDGFR inhibitors with a unique positioning of a single side chain also inhibited CDK8/19 and stimulated phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Marc Paradise, Ryan Shepard, Judith Varner. In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1669.

Dual suppression of follicle activation pathways completely prevents the cyclophosphamide-induced loss of ovarian reserve.

Dual suppression of follicle activation pathways completely prevents the cyclophosphamide-induced loss of ovarian reserve.

Abstract 419: Elevated Akt/mTOR signaling drives therapy resistance in ALM

Abstract Metastatic acral lentiginous melanoma (ALM) is the most lethal subtype of cutaneous melanoma and effective therapies for patients have not been established. Studies revealed a link between the dysregulation of CDK4/6 and ALM mortality, however CDK4/6 inhibitors (CDK4i/6i) have proved ineffective in patients with advanced ALM. The tumor suppressor Rb was recently shown to have a novel suppressive role towards mTORC2. Here, we elucidate the role of a non-canonical tumor suppressor function of Rb in negative regulation of mTOR in CDK4i/6i-resistance. We hypothesize that the loss of Rb induced by CDK4i/6i triggers phosphorylation of Akt and downstream mTORC1 signaling pathways. We characterized baseline activity of the PI3K/AKT/mTOR pathway in a panel of ALM cell lines, finding heterogeneity across signaling. We immunoblotted for key proteins involved in the signal reprogramming hypothesized: pRb, pAkt, pS6 to define activation status and found the loss of Rb phosphorylation preceded the enrichment of pAKT, supporting the non-canonical role of Rb tumor suppression and the Rb-mTORC2 signaling axis as intrinsic CDK4i/6i mechanism. In parallel, we assessed the essential protein-protein interactions required for mechanistic mTORC1 activation—mTOR and Rheb GTPase. The enrichment of these interactions shortly after introducing CDK4i/6i supported our hypothesized mechanism. Translational utility of CDK4i/6i and mTORi dual inhibition was determined via long-term Colony Formation Assays (CFAs) using Palbociclib, rapamycin and sapanisertib (TAK228), a catalytic mTOR inhibitor. We generated Palbociclib (acquired)-resistant ALM cell lines and investigated the pharmacological inhibition of PI3K signaling pathways through immunoblot and colony-formation assays. Concurrent mTORi and CDK4i/6i inhibition showed effective reduction in pRb, pS6, Cyclin D and increases in Cleaved PARP protein, suggesting apoptotic cell death was elevated with combined CDK4i/6i+mTORi treatment. Similarly, CFAs revealed dual inhibition elicited superior antiproliferative effect over the monotherapy treatments. CFAs with acquired-resistance cell lines also showed that combination inhibition decreased colony formation, showing the potential utility of concurrent CDK4i/6i and mTORi as an approach against acquired resistance. Our results show the efficacy of combined inhibition as an anti-tumor strategy. Citation Format: Jeremy A. Bravo Narula, Vito W. Rebecca. Elevated Akt/mTOR signaling drives therapy resistance in ALM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 419.

Phosphatidylinositol 3-kinase signaling pathway and inflammatory bowel disease: Current status and future prospects.

Inflammatory bowel disease (IBD) is a chronic life-limiting disease of gastrointestinal tract characterized by widespread enteric inflammation. IBD is a multifactorial disease, and different environmental, microbial, and immune-related factors give rise to the development of disease. Among several factors, the preponderance of pro-inflammatory T helper 17 cells over the anti-inflammatory regulatory T cells augments inflammation in the intestinal mucosa. Prevailing evidence accentuates that PI3K signaling pathway plays a central role in the pathophysiology of the condition by regulating the inflammatory process in the gut mucosa. By recognizing the implications of PI3K in the pathogenesis of IBD, agents that could modulate this pathway have recently been at the focus of research, yielding encouraging results mainly in the experimental IBD models. In this review, we have summarized the recent advances, which may hold the keys to identify novel therapeutic strategies for IBD.

Comparing gene expression in deep infiltrating endometriosis with adenomyosis uteri: evidence for dysregulation of oncogene pathways

BackgroundThe pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a benign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is not clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different biological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures of both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide clues to pathomechanisms of tumor development based on these diseases.MethodsIn this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent cohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients with histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and RNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer (Nanostring Technology).ResultsIn total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in DIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and KEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes upregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, on the other hand, genes of the RAS pathway showed significant expression compared to DIE.ConclusionDIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those belonging to the PI3K pathway, and in FA those belonging to the RAS pathway.

Inhibition of phosphoinositide‑3 kinases γ/δ ameliorates pulmonary granuloma by rescuing Treg function in a sarcoidosis model.

Sarcoidosis is a multisystem inflammatory disease characterized by the development of Th1/Th17/regulatory T cells (Tregs)-related non-caseating granulomas. Phosphoinositide-3 kinases δ/γ (PI3Kδ/γ) play an important role in the maintenance of effective immunity, especially for Tregs homeostasis and stability. In the present study, superoxide dismutase A (SodA) stimulation was used to establish the sarcoidosis mouse model. The second immune stimulus was accompanied by CAL-101 (PI3Kδ inhibitor) or AS-605240 (PI3Kδ/γ inhibitor) treatment. To detect the effect of the PI3Kδ/γ inhibitor on the morphology of pulmonary granuloma and the activation of the PI3K signaling pathway, hematoxylin and eosin staining and immunofluorescence and western blotting was used, respectively. Fluorescence-activated cell sorting analysis and reverse transcription-quantitative PCR were adopted to detect the effect of the PI3Kδ/γ inhibitor on the SodA-induced sarcoidosis mouse model in respect to immune cell disorder and the function of Treg cells, with CD4+CD25- T cells and CD4+CD25+ T cells sorted by magnetic cell sorting. The results demonstrated that the inhibition of PI3Kδ/γ by transtracheal CAL-101/AS-605240 administration facilitated pulmonary granuloma formation. These therapeutic effects were associated with certain mechanisms, including suppressing the aberrantly activated PI3K/Akt signaling in both pulmonary granuloma and Tregs, particularly rescuing the suppressive function of Tregs. Notably, CAL-101 was more effective in immune modulation compared with AS-605240 and could overcome the aberrantly activated Akt in the lung and Tregs. These results suggest that PI3K/Akt signaling, especially the PI3Kδ subunit, can play a key role in optimal Tregs-mediated protection against pulmonary sarcoidosis. Therefore, transtracheal usage of PI3Kδ/γ inhibitors is an attractive therapy that may be developed into a new immune-therapeutic principle for sarcoidosis in the future.

HMGA1 augments palbociclib efficacy via PI3K/mTOR signaling in intrahepatic cholangiocarcinoma

BackgroundIntrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer that is challenging to diagnose at an early stage. Despite recent advances in combination chemotherapy, drug resistance limits the therapeutic value of this regimen. iCCA reportedly harbors high HMGA1 expression and pathway alterations, especially hyperactivation of the CCND1/CDK4/CDK6 and PI3K signaling pathway. In this study, we explored the potential of targeting CDK4/6 and PI3K inhibition to treat iCCA.MethodsThe significance of HMGA1 in iCCA was investigated with in vitro/vivo experiments. Western blot, qPCR, dual-luciferase reporter and immunofluorescence assays were performed to examine the mechanism of HMGA1 induced CCND1 expression. CCK-8, western blot, transwell, 3D sphere formation and colony formation assays were conducted to predict the potential role of CDK4/6 inhibitors PI3K/mTOR inhibitors in iCCA treatment. Xenograft mouse models were also used to determine the efficacy of combination treatment strategies related to HMGA1 in iCCA.ResultsHMGA1 promoted the proliferation, epithelial-mesenchymaltransition (EMT), metastasis and stemness of iCCA. In vitro studies showed that HMGA1 induced CCND1 expression via promoting CCND1 transcription and activating the PI3K signaling pathway. Palbociclib(CDK4/6 inhibitor) could suppress iCCA proliferation, migration and invasion, especially during the first 3 days. Although there was more stable attenuation of growth in the HIBEpic model, we observed substantial outgrowth in each hepatobiliary cancer cell model. PF-04691502(PI3K/mTOR inhibitor) exhibited similar effects to palbociclib. Compared with monotherapy, the combination retained effective inhibition for iCCA through the more potent and steady inhibition of CCND1, CDK4/6 and PI3K pathway. Furthermore, more significant inhibition of the common downstream signaling pathways is observed with the combination compared to monotherapy.ConclusionsOur study reveals the potential therapeutic role of dual inhibition of CDK4/6 and PI3K/mTOR pathways in iCCA, and proposes a new paradigm for the clinical treatment of iCCA.

Exploring the putative role of PRDM1 and PRDM2 transcripts as mediators of T lymphocyte activation

BackgroundT cell activation and programming from their naïve/resting state, characterized by widespread modifications in chromatin accessibility triggering extensive changes in transcriptional programs, is orchestrated by several cytokines and transcription regulators. PRDM1 and PRDM2 encode for proteins with PR/SET and zinc finger domains that control several biological processes, including cell differentiation, through epigenetic regulation of gene expression. Different transcripts leading to main protein isoforms with (PR +) or without (PR-) the PR/SET domain have been described. Although many studies have established the critical PRDM1 role in hematopoietic cell differentiation, maintenance and/or function, the single transcript contribution has not been investigated before. Otherwise, very few evidence is currently available on PRDM2. Here, we aimed to analyze the role of PRDM1 and PRDM2 different transcripts as mediators of T lymphocyte activation.MethodsWe analyzed the transcription signature of the main variants from PRDM1 (BLIMP1a and BLIMP1b) and PRDM2 (RIZ1 and RIZ2) genes, in human T lymphocytes and Jurkat cells overexpressing PRDM2 cDNAs following activation through different signals.ResultsT lymphocyte activation induced an early increase of RIZ2 and RIZ1 followed by BLIMP1b increase and finally by BLIMP1a increase. The “first” and the “second” signals shifted the balance towards the PR- forms for both genes. Interestingly, the PI3K signaling pathway modulated the RIZ1/RIZ2 ratio in favor of RIZ1 while the balance versus RIZ2 was promoted by MAPK pathway. Cytokines mediating different Jak/Stat signaling pathways (third signal) early modulated the expression of PRDM1 and PRDM2 and the relationship of their different transcripts confirming the early increase of the PR- transcripts. Different responses of T cell subpopulations were also observed. Jurkat cells showed that the acute transient RIZ2 increase promoted the balancing of PRDM1 forms towards BLIMP1b. The stable forced expression of RIZ1 or RIZ2 induced a significant variation in the expression of key transcription factors involved in T lymphocyte differentiation. The BLIMP1a/b balance shifted in favor of BLIMP1a in RIZ1-overexpressing cells and of BLIMP1b in RIZ2-overexpressing cells.ConclusionsThis study provides the first characterization of PRDM2 in T-lymphocyte activation/differentiation and novel insights on PRDM1 and PRDM2 transcription regulation during initial activation phases.

DICER1-associated central nervous system sarcoma: A comprehensive clinical and genomic characterization of case series of young adult patients.

DICER1 alterations are associated with intracranial tumors in the pediatric population, including pineoblastoma, pituitary blastoma, and the recently described "primary DICER1-associated CNS sarcoma" (DCS). DCS is an extremely aggressive tumor with a distinct methylation signature and a high frequency of co-occurring mutations. However, little is known about its treatment approach and the genomic changes occurring after exposure to chemoradiotherapy. We collected clinical, histological, and molecular data from eight young adults with DCS. Genomic analysis was performed by Next-generation Sequencing (NGS). Subsequently, an additional germline variants analysis was completed. In addition, an NGS analysis on post-progression tumor tissue or liquid biopsy was performed when available. Multiple clinicopathological characteristics, treatment variables, and survival outcomes were assessed. Median age was 20 years. Most lesions were supratentorial. Histology was classified as fusiform cell sarcomas (50%), undifferentiated (unclassified) sarcoma (37.5%), and chondrosarcoma (12.5%). Germline pathogenic DICER1 variants were present in two patients, 75% of cases had more than one somatic alteration in DICER1, and the most frequent commutation was TP53. Seven patients were treated with surgery, Ifosfamide, Cisplatin, and Etoposide (ICE) chemotherapy and radiotherapy. The objective response was 75%, and the median time to progression (TTP) was 14.5 months. At progression, the most common mutations were in KRAS and NF1. Overall survival was 30.8 months. DCS is an aggressive tumor with limited therapeutic options that requires a comprehensive diagnostic approach, including molecular characterization. Most cases had mutations in TP53, NF1, and PTEN, and most alterations at progression were related to MAPK, RAS and PI3K signaling pathways.

Regulatory Role of Insulin on Endogenous L1 ORF1 and NEFM Gene Expression through PI3K Signaling Pathway Specifically in Neuroblastoma Cell Line.

One of the most important endogenous factors causing genomic instability in human cells is L1s retrotransposons. In this study, we assume that increased activity of L1 retrotransposons (specifically L1 expression) might be induced by hyperglycemia and hyperinsulinemia in neuroblastoma cell line. Two different cell lines (BE (2)-M17 and HEK293) were treated with insulin and its PI3K signaling pathway inhibitor under three conditional media including hyperglycemic and retinoic acid treatment in the department of Medical Genetics, Tehran University of Medical Sciences, Tehran, Iran in 2018. The expression of L1 ORF1, as well as genes involved in insulin signaling pathway and neuronal stress and structure were measured at RNA level. Insulin could significantly down regulate the expression of L1 ORF1 and NEFM genes. Hyperglycemia result in severe decrease in expression of all candidate genes in control neuroblastoma but not HEK293 cells. Retinoic acid as the concentration used in this study cause increase stemness in neuroblastoma but not HEK293 cells. We could not find significant correlation between expression pattern of other genes tested in our study and L1 ORF1 expression. Total regulatory effect of insulin on L1 ORF1 RNA expression as well as NEFM markedly in BE (2)-M17 cell line. Although these results could not be interpreted as L1 retrotransposition, expression of L1 RNA during stress conditions might be considered following inhibition of the insulin pathway. The result of this study also confirms the impotence of insulin on human evolution.