Expression Of Tyrosine Kinase Research Articles

Abstract 2335: Targeting a positive feedback loop of MERTK and STAT3 during macrophage differentiation may provide anti-tumor immune function

Abstract MERTK tyrosine kinase expression is upregulated upon monocyte to macrophage differentiation. This receptor tyrosine kinase enables macrophages to efficiently clear apoptotic cells to maintain tissue homeostasis. Activation of MERTK in macrophages during efferocytosis promotes an immunosuppressive phenotype, which is hijacked by tumors to inhibit anti-tumor immunity. Further, this immunosuppressive phenotype in MERTK expressing macrophages in the tumor microenvironment is reversed using MERTK selective inhibitors, suggesting that inhibiting MERTK expression or activity in the tumor microenvironment may be of therapeutic benefit in the treatment of cancer. In an attempt to further understand the mechanism of MERTK upregulation in macrophages, we treated the monocytic leukemia cell line THP1 with PMA to differentiate the cells from a monocytic morphology to an adherent macrophage-like morphology. Following differentiation of the THP-1 cells, MERTK upregulation was confirmed by western blot and flow cytometry. In a similar fashion, treatment with of murine bone marrow derived monocytic cells with PMA induced MERTK expression. Proteomics cytokine array analysis also revealed increased levels of multiple chemokines including MCP-1 and RANTES. Treatment of THP-1 cells with a pan STAT inhibitor in the presence of PMA abrogated the induction of MERTK expression, suggesting a critical role for STAT pathways in the regulation of MERTK expression during monocyte differentiation to macrophages. Specifically, the STAT3 pathway was found to be important in MERTK regulation, as treatment with a STAT3 selective inhibitor was sufficient to abrogate MERTK expression in the presence of PMA treatment. Single cell sequencing of the immune cells in the bone marrow demonstrated Stat3 expression in monocytic lineage cells. Furthermore, both CD11C+Ly6c+CD45+ population and MERTK+CD11C+Ly6c+CD45+ populations were lower in Stat3 -/- bone marrow cells treated with PMA relative to the untreated cells. Collectively, these data suggest that MERTK expression during macrophage maturation may be mediated by STAT3 activation. Previously published data have also demonstrated that STAT3 can be activated downstream of MERTK activation. Thus, we propose that MERTK and STAT3 form a positive feedback loop during macrophage maturation. Treatment with either a MERTK and/or STAT3 inhibitor may interfere with this feedback pathway, potentially reversing an immunosuppressive phenotype in the macrophages in the tumor microenvironment. Citation Format: Dan Yan, Justus M. Huelse, Swati Sharma Bhasin, Manoj Bhasin, Deborah DeRyckere, Douglas K. Graham. Targeting a positive feedback loop of MERTK and STAT3 during macrophage differentiation may provide anti-tumor immune function [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 2335.

Circulatory and Placental Expression of Soluble Fms-like Tyrosine Kinase- 1 and Placental Growth Factor in HIV-infected Preeclampsia.

An imbalance between angiogenic and anti-angiogenic factors plays a fundamental role in the pathogenesis of preeclampsia (PE). Studies have shown a dysregulation of sFlt-1 and placental growth factor (PlGF) in PE. However, there are differing reports on the levels of these pro-/antiangiogenic factors in HIV-infected preeclamptic and normotensive pregnancies, possibly due to highly active antiretroviral therapy (HAART) and its immune reconstitution effect. The study aimed to investigate the effect of hypertension and ARVs on circulating and placental pro- and antiangiogenic factors in HIV-infected PE. The level of sFlt-1 expression is elevated in PE compared to normal pregnancies. PlGF was altered by placental dysfunction. Antiretroviral therapy does not impact the angiogenic shift in PE development. The angiogenic imbalance evident in the circulatory system by higher sFlt-1 compared to PlGF levels is replicated in the placenta by reduced expression of PlGF receptors in comparison to sFlt-1 receptors. However, there is a lack of data that explore the relationship between HAART and anti-angiogenic factors in the placenta and the circulation of PE comorbid with HIV infection.

456 Analysis of Clinical, Histologic, and Molecular Characteristics of Proliferative Verrucous Leukoplakia

OBJECTIVES/GOALS: This study aims to develop objectively scored histological characteristics of early oral leukoplakia, which may be correlated with molecular pathways predictive of progression into proliferative verrucous leukoplakia (PVL). The secondary aim is to develop a biomarker profile to be used in diagnosis, staging, and management of PVL. METHODS/STUDY POPULATION: Clinical and pathology records of 120 patients with oral leukoplakia and/or PVL were reviewed. Eight patients were selected—all had serial biopsies over time leading to PVL suspicion. Specimens were deidentified and subjected to blinded examination by a board certified oral pathologist, then scored relative to the extent of each of the commonly accepted histologic characteristics of PVL: hyperkeratosis, acanthosis, blunt rete ridges, hyperchromatic nuclei, increased nuclear-cytoplasmic ratio, dyskeratosis, and surface corrugation. Given these results, a larger subset of patient samples will be labeled and assayed for expression of epidermal growth factor receptor tyrosine kinases and downstream pro-oncogenic signaling mediators. Expression of these factors will be tested against progression to PVL. RESULTS/ANTICIPATED RESULTS: Histologically, in scoring the specimens from eight subjects, the characteristics of acanthosis, dyskeratosis, and blunted rete ridges had the strongest correlation with eventual progression to PVL. These criteria will therefore be recommended as an objective histopathologic method of identification of patients with high risk of development of PVL, and therefore malignant potential. We expect the results of the biomarker assay to provide a molecular basis for predicting PVL pathogenesis. Particularly, we anticipate pro-oncogenic targets such as EGFR, PI3K, Akt, and mTOR pathways will show increased expression as leukoplakic lesions progress. These results would then provide the basis for testing patient samples for expression of these markers in a longitudinal study of PVL emergence and progression. DISCUSSION/SIGNIFICANCE: The aggressive nature of PVL, with a rate of malignant transformation of 61% and mortality rate of 40%, requires close clinical monitoring in order to improve patient outcomes. Therefore, well defined objective clinical, histologic, and molecular criteria are critical for early detection of sites likely to progress to PVL and subsequent malignancy.

HZ-A-018, a novel inhibitor of Bruton tyrosine kinase, exerts anti-cancer activity and sensitizes 5-FU in gastric cancer cells.

Gastric cancer is the third leading cause of cancer related death worldwide. Due to the complexity and heterogeneity of gastric cancer, the development of targeted drugs is somehow limited, but is urgently needed. Since the expression of Bruton tyrosine kinase (BTK) was significantly associated with the prognosis of gastric cancer patients, we aimed to determine the anti-cancer activity of HZ-A-018, which was a novel derivative of ACP-196, in gastric cancer cells. As a result, HZ-A-018 presented a stronger anti-proliferation activity than ACP-196 via the substantial suppression of AKT/S6 pathway. In addition, HZ-A-018, but not ACP-196, exerted the synergistic effects in combined treatment with 5-FU both in vitro and in vivo, without exacerbating the adverse effects of 5-FU. Mechanismly, the combination of HZ-A-018 and 5-FU remarkably reduced the expression of RRM2, which played an essential role in proliferation and drug sensitivity in gastric cancer cells. In summary, our work demonstrated the stronger anti-cancer activity of HZ-A-018 than ACP-196 in gastric cancer cells, and revealed synergistic effects of HZ-A-018 and 5-FU combination probably through the inhibition of RRM2 via AKT/S6 pathway, thereby providing a promising therapeutic strategy in gastric cancer.

Alterations in the Proteome of Developing Neocortical Synaptosomes in the Absence of MET Signaling Revealed by Comparative Proteomics

Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.

Culture Conditions for Mycelial Growth and Anti-Cancer Properties of Termitomyces

Termitomyces sp. that grow in symbiosis with fungus-farming Termites have medicinal properties. However, they are rare in nature, and their artificial culture is challenging. The expression of AXL receptor tyrosine kinase and immune checkpoint molecules favor the growth of cancer cells. The study evaluated the optimal conditions for the artificial culture of Termitomyces and their inhibitory activity on AXL and immune checkpoint molecules in lung adenocarcinoma and melanoma cell lines. The culture of 45 strains of Termitomyces was compared. Five strains with marked growth rates were selected. Four of the selected strains form a single cluster by sequence analysis. The mycelium of 4 selected strains produces more fungal mass in potato dextrose broth than in a mixed media. The bark was the most appropriate solid substrate for Termitomyces mycelia culture. The mycelium of all five selected strains showed a higher growth rate under normal CO2 conditions. The culture broth, methanol, and ethyl acetate of one selected strain (T-120) inhibited the mRNA relative expression of AXL receptor tyrosine kinase and immune checkpoint molecules in cancer cell lines. Overall, these results suggest the potential usefulness of Termitomyces extracts as a co-adjuvant therapy in malignant diseases.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Proteomic quantification of receptor tyrosine kinases involved in the development and progression of colorectal cancer liver metastasis.

Alterations in expression and activity of human receptor tyrosine kinases (RTKs) are associated with cancer progression and in response to therapeutic intervention. Thus, protein abundance of 21 RTKs was assessed in 15 healthy and 18 cancerous liver samples [2 primary and 16 colorectal cancer liver metastasis (CRLM)] matched with non-tumorous (histologically normal) tissue, by a validated QconCAT-based targeted proteomic approach. It was demonstrated, for the first time, that the abundance of EGFR, INSR, VGFR3 and AXL, is lower in tumours relative to livers from healthy individuals whilst the opposite is true for IGF1R. EPHA2 was upregulated in tumour compared with histologically normal tissue surrounding it. PGFRB levels were higher in tumours relative to both histologically normal tissue surrounding tumour and tissues taken from healthy individuals. The abundances of VGFR1/2, PGFRA, KIT, CSF1R, FLT3, FGFR1/3, ERBB2, NTRK2, TIE2, RET, and MET were, however, comparable in all samples. Statistically significant, but moderate correlations were observed (Rs > 0.50, p < 0.05) for EGFR with INSR and KIT. FGFR2 correlated with PGFRA and VGFR1 with NTRK2 in healthy livers. In non-tumorous (histologically normal) tissues from cancer patients, there were correlations between TIE2 and FGFR1, EPHA2 and VGFR3, FGFR3 and PGFRA (p < 0.05). EGFR correlated with INSR, ERBB2, KIT and EGFR, and KIT with AXL and FGFR2. In tumours, CSF1R correlated with AXL, EPHA2 with PGFRA, and NTRK2 with PGFRB and AXL. Sex, liver lobe and body mass index of donors had no impact on the abundance of RTKs, although donor age showed some correlations. RET was the most abundant of these kinases in non-tumorous tissues (~35%), while PGFRB was the most abundant RTK in tumours (~47%). Several correlations were also observed between the abundance of RTKs and proteins relevant to drug pharmacokinetics (enzymes and transporters). DiscussionThis study quantified perturbation to the abundance of several RTKs in cancer and the value generated in this study can be used as input to systems biology models defining liver cancer metastases and biomarkers of its progression.

Prognostic Significance of ARID1A Expression Patterns Varies with Molecular Subtype in Advanced Gastric Cancer.

AT-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancer (GC), especially Epstein-Barr virus (EBV)-associated and microsatellite instability high GC. The loss of ARID1A expression has been reported as a poor prognostic marker in GC. However, the relationships between ARID1A alteration and EBV-associated and microsatellite instability high GC, which are known to have a favorable prognosis, has hampered proper evaluation of the prognostic significance of ARID1A expression in GC. We aimed to analyze the true prognostic significance of ARID1A expression by correcting confounding variables. We evaluated the ARID1A expression in a large series (n=1,032) of advanced GC and analyzed the relationships between expression pattern and variable parameters, including clinicopathologic factors, key molecular features such as EBV-positivity, mismatch repair protein deficiency, and expression of p53 and several receptor tyrosine kinases including human epidermal growth factor receptor 2, epidermal growth factor receptor, and mesenchymal-epithelial transition factor. Survival analysis of the molecular subtypes was done according to the ARID1A expression patterns. Loss of ARID1A expression was found in 52.5% (53/101) of mutL homolog 1 (MLH1)-deficient and 35.8% (24/67) of EBV-positive GCs, compared with only 9.6% (82/864) of the MLH1-proficient and EBV-negative group (p<0.001). The loss of ARID1A expression was associated only with MLH1 deficiency and EBV positivity. On survival analysis, the loss of ARID1A expression was associated with worse prognosis only in MLH1-proficient and EBV-negative GC. Multivariate analysis revealed that both loss of ARID1A and decreased ARID1A expression were independent worse prognostic factors in patients with advanced GC. Only in MLH1-proficient and EBV-negative GC, the loss of ARID1A expression is related to poorer prognosis.

RON Receptor Tyrosine Kinase in Tumorigenic Stemness as a Therapeutic Target of Antibody-drug Conjugates for Eradication of Triple-negative Breast Cancer Stem Cells.

Cancer stem-like cells in triple-negative breast cancer (TNBC-SLCs) are the tumorigenic core for malignancy. Aberrant expression of the RON receptor tyrosine kinase has implications in TNBC tumorigenesis and malignancy. In this study, we identified the RON receptor as a pathogenic factor contributing to TNBC cell stemness and validated anti-RON antibody-drug conjugate Zt/g4-MMAE for eradication of RONexpressing TNBC-SLCs. Immunofluorescence and Western blotting were used for analyzing cellular marker expression. TNBC-SLCs were isolated by magnetic-immunofluorescence cell-sorting techniques. Spheroids were generated using the ultralow adhesion culture methods. Levels of TNBC-SLC chemosensitivity were determined by MTS assays. TNBC-SLC mediated tumor growth was determined in athymic nude mice. The effectiveness of Zt/g4-induced RON internalization was measured by immunofluorescence analysis. Efficacies of Zt/g4-MMAE in killing TNBC-SLCs in vitro and in eradicating TNBC-SLCmediated tumors were determined in mouse models. All data were statistically analyzed using the GraphPad Prism 7 software. Increased RON expression existed in TNBC-SLCs with CD44+/CD24- phenotypes and ALDH activities and facilitated epithelial to mesenchymal transition. RON-positive TNBC-SLCs enhanced spheroid-formatting capability compared to RON-negative TNBC-SLCs, which were sensitive to small molecule kinase inhibitor BMS-777607. Increased RON expression also promoted TNBC-SLC chemoresistance and facilitated tumor growth at an accelerated rate. In vitro, Zt/g4-MMAE caused massive TNBC-SLC death with an average IC50 value of ~1.56 μg per/ml and impaired TNBC cell spheroid formation. In mice, Zt/g4-MMAE effectively inhibited and/or eradicated TNBC-SLC mediated tumors in a single agent regimen. Sustained RON expression contributes to TNBC-SLC tumorigenesis. Zt/g4-MMAE is found to be effective in vivo in killing TNBC-SLC-mediated xenograft tumors. Our findings highlight the feasibility of Zt/g4-MMAE for the eradication of TNBC-SLCs in the future.

Cyclohexane-1,3-dione Derivatives as Future Therapeutic Agents for NSCLC: QSAR Modeling, In Silico ADME-Tox Properties, and Structure-Based Drug Designing Approach.

The abnormal expression of the c-Met tyrosine kinase has been linked to the proliferation of several human cancer cell lines, including non-small-cell lung cancer (NSCLC). In this context, the identification of new c-Met inhibitors based on heterocyclic small molecules could pave the way for the development of a new cancer therapeutic pathway. Using multiple linear regression (MLR)-quantitative structure-activity relationship (QSAR) and artificial neural network (ANN)-QSAR modeling techniques, we look at the quantitative relationship between the biological inhibitory activity of 40 small molecules derived from cyclohexane-1,3-dione and their topological, physicochemical, and electronic properties against NSCLC cells. In this regard, screening methods based on QSAR modeling with density-functional theory (DFT) computations, in silico pharmacokinetic/pharmacodynamic (ADME-Tox) modeling, and molecular docking with molecular electrostatic potential (MEP) and molecular mechanics-generalized Born surface area (MM-GBSA) computations were used. Using physicochemical (stretch-bend, hydrogen bond acceptor, Connolly molecular area, polar surface area, total connectivity) and electronic (total energy, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels) molecular descriptors, compound 6d is identified as the optimal scaffold for drug design based on in silico screening tests. The computer-aided modeling developed in this study allowed us to design, optimize, and screen a new class of 36 small molecules based on cyclohexane-1,3-dione as potential c-Met inhibitors against NSCLC cell growth. The in silico rational drug design approach used in this study led to the identification of nine lead compounds for NSCLC therapy via c-Met protein targeting. Finally, the findings are validated using a 100 ns series of molecular dynamics simulations in an aqueous environment on c-Met free and complexed with samples of the proposed lead compounds and Foretinib drug.

Abstract A036: Juxtacrine signaling between macrophage and cancer cell inhibits radiation-induced ferroptosis in syngeneic colon cancer

Abstract Although radiotherapy (RT) is one of the most common modalities for cancer treatment, radioresistance remains an intractable challenge. RT induces intratumoral infiltration of macrophages for the clearance of injury debris, but rather tumor-associated macrophages confer fitness advantages to cancer cells after RT. Here, we report that tumor-associated macrophage instigates radioresistance by inhibiting RT-induced cell death in a contact-dependent manner. Mechanistically, RT increases receptor tyrosine kinases (RTKs) expression in cancer cells to enable direct interaction with macrophages, resulting in reduced cancer cell ferroptosis triggered by RT. Unbiased RNA-seq analysis of RTK-dependent juxtacrine signaling in macrophages further reveals a profound remodeling of the irradiated tumor microenvironment (TME) via regulation of macrophage polarization and cytotoxic T cell-mediated antitumor responses. Using a syngeneic colon cancer model, we found that ablation of RTK-induced juxtacrine signaling sensitized tumor to RT through increasing cancer cell ferroptosis and impairing macrophages-mediated TME. Collectively, our study supports the RTKs as a new radiosensitizing target via attenuation of the interaction between cancer cells and macrophages during RT. Citation Format: Sang Wha Kim, Hyewon Chung, Seung Hyeok Seok. Juxtacrine signaling between macrophage and cancer cell inhibits radiation-induced ferroptosis in syngeneic colon cancer [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A036.

Warm acupuncture stimulation improves cartilage damage and motor function by regulating JAK2/STAT3 signaling pathway in rabbits with knee osteoarthritis

To observe the effect of warm acupuncture on the expression of Janus protein tyrosine kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway and inflammatory factors of articular cartilage in rabbits with knee osteoarthritis (KOA), so as to explore its underlying mechanisms in improving KOA. New Zea-land rabbits were randomly divided into blank, model, warm acupuncture and medication groups (12 rabbits in each group). The KOA model was prepared by using the right hind limb tubular plaster extension fixation method. The rabbits in the warm acupuncture group received acupuncture of "Neixiyan"(EX-LE4),"Waixiyan"(ST35),"Heding"(EX-LE2) and "Zusanli"(ST36), followed by attaching an ignited moxa-stick segment to the acupuncture-handle. The treatment was conducted for 15 min, once a week for 4 weeks. The rabbits in the medication group received gavage of diclofenac sodium solution(0.35 mg/kg), once daily for 4 weeks. The dysfunction severity state of the rabbit's knee-joint was evaluated using Lequesne scale (0-3 points), and the histopathological changes of cartilage were observed under microscope after H.E. staining and the state of distribution of chondrocytes in different layers and the extracellular matrix was assessed using Mankin score (0-6 points). The contents of serum interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) were measured by using ELISA, and the expression levels of p-JAK2/JAK2, p-STAT3/STAT3 and MMP-9 in knee cartilage tissue were detected using Western blot. Compared with the blank group, the Lequesne score, Mankin score, and the contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression level of MMP-9 protein in knee cartilage tissue were significantly increased in the model group (P<0.01). In comparison with the model group, the Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, and the expression of MMP-9 protein in knee cartilage tissue were notably decreased in both the warm acupuncture and medication groups (P<0.01,P<0.05). The levels of Lequesne score, Mankin score, contents of serum IL-6, TNF-α and MMP-9, and the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 in knee cartilage tissue were significantly lower in the warm acupuncture group than in the medication group (P<0.01, P<0.05). No significant difference was found between the warm acupuncture and medication groups in the expression of MMP-9 protein (P>0.05). Outcomes of H.E. showed injury of the perichondrium of knee joint, obvious reduction of the cartilage matrix staining, cystic changes, clustered and disordered arrangement and severe pyknosis and necrosis of the surface cells with reduction of number of cells and increase of vacuoles in the model group, which was milder in both warm acupuncture and medication groups. Warm acupuncture can improve motor function and reduce cartilage injury in KOA rabbits, which may be related to its functions in inhibiting the secretion of pro-inflammatory factors and regulating JAK2/STAT3 signaling and downregulating MMP-9 expression in the cartilage tissue.

Reduced expression in preterm birth of sFLT-1 and PlGF with a high sFLT-1/PlGF ratio in extracellular vesicles suggests a potential biomarker.

Preterm birth may have a pathological impact on intrauterine development of the fetal brain, resulting in developmental disabilities. In this study, we examine the expression of soluble Fms-like tyrosine kinase 1 (sFLT-1) and placental growth factor (PlGF), which is one of the vascular endothelial growth factors (VEGFs), as these play a key role in angiogenesis; in particular, we examine their effect on the sFLT-1/PlGF ratio in cases of preterm birth as compared to typical pregnancies. Enzyme-linked immunosorbent assay was performed on samples of maternal-derived plasma and extracellular vesicles-exosomes (EVs-EXs) isolated at the third trimester, consisting of 17 samples from cases of preterm birth and 38 control cases. Our results showed that both sFLT-1 (P=0.0014) and PlGF (P=0.0032) were significantly downregulated in cases of preterm birth compared to controls, while the sFLT-1/PIGF ratio was significantly (P=0.0008) increased in EVs-EXs, but not in maternal plasma. Our results suggest that this reduced expression of sFLT-1 and PlGF with an elevated sFLT-1/PlGF ratio in EVs-EXs may represent a potential biomarker for prediction of PTB.

Upregulation of PIK3IP1 monitors the anti-cancer activity of PI3Kα inhibitors in gastric cancer cells

Gastric cancer remains one of the most malignant cancers in the world. The target-based drugs approved by FDA for gastric cancer treatment include only three targets and benefit a small portion of gastric cancer patients. PIK3CA, a confirmed oncogene, mutates in 7–25% gastric cancer patients. PI3Kα inhibitor BYL719 has been approved for treating specific breast cancer. However, there is no comprehensive study about PI3Kα inhibitor in gastric cancer. In this study, we found pharmacological inhibition or knockdown of PI3Kα effectively inhibited the proliferation of partial gastric cancer cells. Then, we systematically explored the potential biomarkers for predicting or monitoring treatment response according to previous reports and found that basal expression of several receptor tyrosine kinases were related with the sensitivity of gastric cancer cells to BYL719. Next, RNA-seq technique was utilized and showed that BYL719 inhibited Myc targets V2 gene set in sensitive gastric cancer cells, and western blotting further verified that c-Myc was only inhibited in sensitive gastric cancer cells. More importantly, we firstly found BYL719 significantly elevated the expression of PIK3IP1 in sensitive gastric cancer cells, which was also observed in NCI-N87 cell derived xenograft mice models. Meanwhile, knockdown of PIK3IP1 partially rescued the cell growth inhibited by BYL719 in sensitive gastric cancer cells, suggesting the important role of PIK3IP1 in the antitumor activity of BYL719. In conclusion, our study provides biological evidence that PI3Kα is a promising target in specific gastric cancer and the elevation of PIK3IP1 could supply as a biomarker that monitoring treatment response.

Abstract PR011: Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer

Abstract We have previously identified two tumor specific molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), basal-like and classical. Although basal-like tumors are found in less than 20% of patients, patients with basal-like tumors have a worse prognosis and are largely resistant to FOLFIRINOX chemotherapy. To identify targetable basal-like subtype specific vulnerabilities in PDAC, we utilized Multiplexed kinase Inhibitor Beads and Mass Spectrometry (MIB-MS) to profile the kinome in patient derived xenograft (PDX) models that recapitulate the tumor subtypes found in patients. MIB-MS results show that kinase expression is significantly associated with molecular subtype. Notably, basal-like tumors show increased expression of receptor tyrosine kinases (RTKs) including EGFR, MET, and IGF1R and MAP kinase members MEK2 and BRAF indicating activation of the MAP kinase signaling cascade. These RTKs including EGFR are also differentially expressed in primary patient tumors suggesting the true efficacy of EGFR and other kinase inhibitors in basal-like tumors may have been masked by the larger proportion of patients (&amp;gt;80%) with unresponsive classical tumors. Furthermore, inhibition of MAP kinase signaling with the MEK inhibitor trametinib results in kinome reprogramming exclusive to classical tumors. MIB-MS profiling reveals upregulation of TAOK3 and MEK3/6 indicating activation of p38 MAP kinase signaling as a classical subtype specific compensatory mechanism against MEK inhibition. Overall, these results present actionable basal-like subtype specific kinase targets by defining a novel subtype specific kinome in PDAC. Precision approaches in clinical trials are needed to determine if new and previously thought to be disappointing kinase inhibitors may be efficacious in patients with basal-like tumors. Citation Format: Yi Xu, Ashley B. Morrison, Silvia G. Herrera, Michael P. East, Gary L. Johnson, Jen Jen Yeh. Proteomic profiling reveals subtype specific kinase expression in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR011.

A Novel BTK Inhibitor Induces Cells Death and Enhances the Efficacies of Venetoclax in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by clonal expansion of immature blast cells in the peripheral blood and bone marrow. AML accounts for nearly 80% of adult leukemia cases. Importantly, receptor tyrosine kinases mutations, as well as high expression of Bruton's tyrosine kinase (BTK) have been identified in AML. BTK is responsible for regulating cell survival and activation through the B-cell receptor signaling pathway, as well as tumor cell adhesion to bone marrow stromal cells. Additionally, anti-apoptotic proteins, especially B-cell lymphoma 2 (BCL-2), are involved in the survival and resistance of AML cells. BCL-2 is overexpressed and is associated with a higher rate of relapse and a decreased sensitivity to chemotherapy. Patients treated with venetoclax, a selective BCL-2 inhibitor, have shown chemotherapeutic resistance in hematologic malignancies, including AML. Drug resistance gradually develops through increasing the expression of BTK and other anti-apoptotic proteins. However, combination therapy can be an effective treatment option for patients to achieve durable chemotherapeutic responses. Multiple combination strategies in the clinic suggest the use of venetoclax with well-established agents in order to increase venetoclax efficacy towards AML and overcome resistance challenges. To identify new therapies for AML, we proposed the use of a novel BTK inhibitor, KS151, alone and in combination with venetoclax to provide an alternative strategy for patients. Interestingly, KS151, a novel BTK inhibitor, induces AML cell death with an average IC50 of 4 μM. Moreover, KS151 was able to increase the efficacy of venetoclax towards AML cells. In our panel of three AML cell lines: Kusami-1, U937, and THP-1, our western blot showed Kusami-1 had the highest expression of BTK and BCL-2 proteins. Remarkably, in a dose-dependent manner, KS151 induces Kusami-1 cell death by targeting BTK and enhances the downregulation of BCL-2 in combination with venetoclax. Annexin V staining in flow cytometry also demonstrated KS151 in combination therapy with venetoclax resulted in apoptosis in nearly 95% of AML cells. Taken together, our findings propose KS151 alone or in combination with venetoclax as a promising potential AML killer and may represent a new viable treatment. Future studies will uncover the mechanism of action of KS151 and include the assessment in patient samples and in vivo studies.

Escape from Type II JAK2 Inhibition in MPN Depends on AXL/MAPK Activation and Is Targetable

Myeloproliferative neoplasms (MPN) are characterized by dysregulated JAK2 signaling. Clinical type I JAK2 inhibitors as ruxolitinib are hampered by limited disease-modifying effects and occurrence of resistance. Based on pre-clinical data, novel type II JAK2 inhibitors as CHZ868 effectively stabilize inactive JAK2, reduce MPN clones and overcome resistance to ruxolitinib. We study whether MPN cells could escape from type ll JAK2 inhibition, and characterize therapeutic approaches. To assess potential escape from type II JAK inhibition, JAK2V617F MPN cells incl. SET2, UKE-1 and Ba/F3 EPOR Jak2V617F were exposed long-term to CHZ868. Outgrowing cells showed significantly increased IC50 to CHZ868 and reduced susceptibility for apoptosis induction. No second-site mutations in JAK2 were detected. Phosphoproteomic analysis identified >2500 differentially phosphorylated sites with most prominent activation of the MAPK pathway in JAK inhibitor resistant cells. Type II JAK inhibition persistently suppressed JAK2 - STAT3/5 signaling, while MAPK target activation incl. MEK and ERK was prominent also by phospho-immunoblot suggesting MAPK pathway signaling as a driver of escape from type II JAK inhibition. To characterize the molecular basis, we profiled differential gene expression and chromatin accessibility by RNA-sequencing and ATAC-sequencing in MPN cells escaping JAK inhibition. Paired analysis of RNA-seq/ATAC-seq revealed enrichment of H3K27me3 and PRC2 signatures with overall more compacted chromatin. Expression of AXL tyrosine kinase known to signal through the MAPK pathway, was significantly increased along with enriched RAS signaling target gene patterns by GSEA. Motif analysis suggested differential transcriptional programs with loss of ETS and increased accessibility of GATA transcription factor family binding sites. Enhanced AXL expression was confirmed by immunoblotting and depletion of AXL by AXL-specific shRNA knock-down resensitized resistant cells to CHZ868 with decreased IC50. Since these findings pointed towards a role of AXL activation for escape of MPN cells from type II JAK inhibition, we targeted AXL activation using respective inhibitors. AXL inhibitor bemcentinib and AXL/FLT3 inhibitor gilteritinib reduced proliferation of resistant cells with up to 5.6-fold and 3.4-fold decreased IC50, respectively. Given that type II JAK inhibition with CHZ868 maintained suppression of JAK2-STAT3/5 activation in resistant cells, we combined JAK2 and AXL inhibition. Notably, dual JAK2 / AXL inhibition with CHZ868 / bemcentinib or CHZ868 / gilteritinib showed most pronounced inhibitory effects with IC50 similar to values seen in JAK inhibitor sensitive cells and effective induction of apoptosis. Resistant MPN cells engrafted subcutaneously into immunocompromised NSG mice and resulted in robust tumor growth despite JAK2 inhibitor treatment. However, AXL inhibition with bemcentinib reduced progression of type II JAK inhibitor resistant tumors. Combined JAK2 and AXL inhibition showed most pronounced suppression of subcutaneous tumor growth with minimal progression. Given that inhibitors of MAPK signaling targeting e.g. MEK-ERK activation, are in clinical use for other malignancies with favorable tolerability profiles, we then characterized combined JAK2 / MAPK inhibition to overcome AXL-MAPK driven escape of MPN cells from type II JAK inhibitors. Combination of CHZ868 and MEK inhibitor trametinib significantly reduced subcutaneous tumor growth in NSG mice similarly to JAK2 / AXL inhibition. Expression of MAPK pathway effectors as DUSP6 was substantially reduced. Furthermore, intravenous engraftment of JAK inhibitor resistant cells into NSG mice led to substantial bone marrow (BM) infiltration shown by luciferase imaging and hCD45 immunohistochemistry despite treatment with CHZ868. Combined JAK2 / MAPK inhibition with CHZ868 and trametinib significantly reduced BM infiltration by luciferase imaging and histology supporting that escape from type II JAK inhibition is dependent on AXL-MAPK activation. In summary, we report on a novel mechanism of AXL-MAPK driven escape from type II JAK2 inhibition which is targetable in vitro and in vivo. Our findings highlight that rationally designed combination therapy approaches can restore sensitivity to JAK inhibitor therapy in MPN, which may have potential implications for other malignant settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-32. SEQUENTIAL SUNITINIB AND AURANOFIN TREATMENT INDUCEDCYTOTOXICITY THROUGH ROS-MEDIATED MECHANISM IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) displays aberrant expression of several receptor tyrosine kinases (RTKs) leading to worse prognosis. Despite aggressive treatment including surgery, radiation and the alkylating agent temozolomide (TMZ), GBM tumors counteract deleterious effects of treatment-induced reactive oxygen species (ROS) leading to resistance to standard treatment. Likewise, expression of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) plays a key role in resistance to TMZ.We previously reported a potential link between MGMT expression and decreased angiogenesis in GBM. The differential expression of MGMT affected response to sunitinib, a multitargeted RTK inhibitor with anti-angiogenic properties FDA-approved in different cancer types. Notably, TMZ-resistant GBM MGMT-positive cells were more vulnerable to sunitinib treatment compared to isogenic MGMT-negative cells. We further provided evidence for a positive relationship between MGMT and the antioxidant enzyme, thioredoxin reductase 1 (TrxR1) in GBM. The TrxR1-targeting drug Auranofin, FDA-approved for rheumatoid arthritis generated ROS and induced more cytotoxic effects in GBM cells displaying low expression of MGMT and TrXR1. We hypothesized that sunitinib-induced anti-proliferative effects might sensitize GBM cells to Auranofin. We showed that MGMT-negative GBM cells, which displayed lower levels of TrxR1, were more vulnerable to Auranofin than MGMT-positive cells. The ROS scavenger N-acetylcysteine (NAC) reverted the cytotoxicity of Auranofin suggesting ROS-mediated cytotoxic effects. Conversely, Sunitinib exhibited ROS-independent anti-proliferative effects in MGMT-positive cells. Remarkably, sequential treatment using sunitinib pre-treatment (2 hours) followed by Auranofin (24 hours) significantly decreased cell viability, clonogenicity, increased ROS, decreased TrXR1 and MGMT expression and sensitized MGMT-positive GBM cells resistant to Auranofin. NAC reverted these synergistic effects, suggesting ROS-mediated mechanism. Our study provides new insights into the modulation of the cellular redox homeostasis using sequential sunitinib and Auranofin. This will enable repurposing Auranofin and sunitinib, two drugs with a known safety profile for an effective therapeutic strategy for GBM patients.

Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

Type I IFN is made by cells in response to stress. Cancer cells exist in a state of stress, but their IFN response is complex and not completely understood. This study investigated the role of autocrine IFN in human chronic lymphocytic leukemia (CLL) cells. CLL cells were found to make low amounts of IFN via TANK-binding kinase 1 pathways, but p-STAT1 and -STAT2 proteins along with IFN-stimulated genes that reflect IFN activation were variably downregulated in cultured CLL cells by the neutralizing IFNAR1 Ab anifrolumab. Patients with CLL were segregated into two groups based on the response of their leukemia cells to anifrolumab. Samples associated with more aggressive clinical behavior indicated by unmutated IGHV genes along with high CD38 and p-Bruton's tyrosine kinase expression exhibited responses to low amounts of IFN that were blocked by anifrolumab. Samples with more indolent behavior were unaffected by anifrolumab. Hypersensitivity to IFN was associated with higher expression of IFNAR1, MX1, STAT1, and STAT2 proteins and lower activity of negative regulatory tyrosine phosphatases. Autocrine IFN protected responsive CLL cells from stressful tissue culture environments and therapeutic drugs such as ibrutinib and venetoclax in vitro, in part by upregulating Mcl-1 expression. These findings suggest hypersensitivity to IFN may promote aggressive clinical behavior. Specific blockade of IFN signaling may improve outcomes for patients with CLL with higher-risk disease.