Citations

27 Background: Cholangiocarcinoma (CCA) is a devastating cancer with few effective treatment options. We have previously discovered LCK can activate AXL, a TAM receptor tyrosine kinase (RTK), by phosphorylation of AXL-Y866 ( J Hepatol 2022). AXL is known to confer therapeutic resistance to solid cancers. However, the role of AXL in CCA, and the impact of AXL-Y866 activation by LCK still remains to be elucidated. Here, we investigated the significance of AXL expression, signaling, and activation by LCK on Y866. Methods: We first evaluated the expression levels of AXL in CCA and its association with patient outcome using The Cancer Genome Atlas (TCGA) dataset. Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GC), AXL downregulation was achieved via siRNA approach and the selective AXL inhibitor bemcentinib (Bem) using human CCA cell lines, HuCCT1 and RBE, and murine SB-1. We examined 50% inhibitory concentration (IC50) value on GC with or without AXL knockdown (KD) using cell viability assay. Then we assessed the efficacy of the combination of GC and Bem utilizing Calcusyn software and apoptosis by Annexin V assay. I n vivo efficacy was assessed using both a patient derived CCA xenograft and SB-1 a syngeneic model of CCA treated with vehicle, GC, Bem, or the combination. Next, we defined the interactome of wildtype AXL- Y866 compared to AXL-Y866F using biotin proximity labelling methods (BioID), validation of downstream target proteins using Western blot (WB), and a functional study with AXL-/- cells reconstituted with either AXL WT, or AXL Y866F. Results: In TCGA cohorts, AXL transcripts are more abundant than in normal adjacent liver, and higher levels are correlated with worse clinical outcome. In in vitro study, IC50 values of GC decreased after AXL KD. Synergistic effects were observed in the combination. The combination caused increased apoptosis compared to other treatments. In the in vivo studies, the combination suppressed tumor growth and the Ki67 levels, and the phosphorylation levels of PEAK1 decreased in the combination group compared to other groups. In BioID experiments, among the 222 proteins which were detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was most altered when Y866 was inactivated by the Y866F mutation. In gene ontology analysis, cell-cell adhesion and focal adhesion related proteins were enriched. In WB, we validated that the phosphorylation of PEAK1 decreased after AXL or LCK knockout (KO) and after Bem treatment in above in vivo study. In cell viability assay, reconstitution of AXL KO cells with WT AXL increased the IC50 to GC while reconstitution with AXL-Y866F did not. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in preclinical model. LCK-AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA.

Lipids play crucial biological roles in health and disease, including in cancers. The phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pivotal promoter of cell growth and proliferation in various types of cancer. The somatic mutations in PIK3CA , the gene coding for the catalytic subunit p110α of PI3K, are frequently present in cancer cells, including breast cancer. Although the most prominent mutants, represented by single amino acid substitutions in the helical domain in exon 9 (E545K) and the kinase domain in exon 20 (H1047R) are known to cause a gain of PI3K function, activate AKT signaling and induce oncogenic transformation, the effect of these mutations on cellular lipid profiles has not been studied. We carried out untargeted lipidomics using liquid chromatography-tandem mass spectrometry to detect the lipid alterations in mammary gland epithelial MCF10A cells with isogenic knockin of these mutations. A total of 536 species of lipids were analyzed. We found that the levels of monosialogangliosides, signaling molecules known to enhance cell motility through PI3K/AKT pathway, were significantly higher in both mutants. In addition, triglycerides and ceramides, lipid molecules known to be involved in promoting lipid droplet production, cancer cell migration and invasion, were increased, whereas lysophosphatidylcholines and phosphatidylcholines that are known to inhibit cancer cell motility were decreased in both mutants. Our results provide novel insights into a potential link between altered lipid profile and carcinogenesis caused by the PIK3CA hotspot mutations. In addition, we suggest untargeted lipidomics offers prospects for precision/personalized medicine by unpacking new molecular substrates of cancer biology.

Objective: The objective of this study was to compare endovascular aortic aneurysm repair (EVAR) versus open aortic repair (OAR) on mortality and reintervention after ruptured infrarenal abdominal aortic aneurysm (rAAA) repair in the Vascular Quality Initiative (VQI). Background: The optimal treatment modality for rAAA remains debated, with little data on long-term comparisons. Methods: VQI rAAA repairs (2004–2018) were matched with Medicare claims (VQI-VISION). Primary outcomes were in-hospital and long-term mortality. Secondary outcome was reintervention. Inverse probability weighting was used to adjust for treatment selection, and Cox Proportional Hazards models and negative binomial regressions were used for analysis. Landmark analysis was performed among patients surviving hospital discharge. Results: Among 1885 VQI/Medicare rAAA patients, 790 underwent OAR, and 1095 underwent EVAR. Median age was 76 years; 73% were male. Inverse probability weighting produced comparable groups. In-hospital mortality was lower after EVAR versus OAR (21% vs 37%, odds ratio: 0.52, 95% CI, 0.4–0.7). One-year mortality rates were lower for EVAR versus OAR [hazard ratio (HR) 0.74, 95% CI, 0.6–0.9], but not statistically different after 1 year (HR: 0.95, 95% CI, 0.8–1.2). This implies additional benefits to EVAR in the short term. Reintervention rates were higher after EVAR than OAR at 2 and 5 years (rate ratio: 1.79 95% CI, 1.2–2.7 and rate ratio:2.03 95% CI, 1.4–3.0), but not within the first year. Reintervention was associated with higher mortality risk for both OAR (HR: 1.66 95% CI, 1.1–2.5) and EVAR (HR: 2.14 95% CI, 1.6–2.9). Long-term mortality was similar between repair types (HR: 0.99, 95% CI, 0.8–1.2). Conclusions: Within VQI/Medicare patients undergoing rAAA repair, the perioperative mortality rate favors EVAR but equalizes after 1 year. Reinterventions were more common after EVAR and were associated with higher mortality regardless of treatment.

Abstract Background: Cholangiocarcinoma (CCA) is a lethal disease with limited therapeutic options. We have previously discovered LCK driving signaling through AXL, a TAM receptor tyrosine kinase (RTK), by phosphoproteomic analysis of CCA (J Hepatol 2022). AXL mediates acquired drug resistance in solid cancers. However, the exact role of AXL in CCA still remains to be elucidated. Here, we investigated the significance of AXL expression as a potential therapeutic target in CCA and the role of phosphorylated AXL Y866. Methods: We first evaluated the expression levels of AXL in CCA and its associations with patient outcomes using The Cancer Genome Atlas (TCGA) database. Next, to evaluate whether AXL inhibition sensitizes CCA cells to gemcitabine and cisplatin (GemCis) therapy, AXL downregulation was achieved via the siRNA approach and the selective inhibitor bemcentinib. We examined the 50% inhibitory concentration (IC50) value of HuCCT1, a well-characterized CCA cell line, on GemCis therapy with or without AXL knockdown using cell viability assay. Then we assessed the efficacy of the combinatorial therapy of GemCis and bemcentinib utilizing Calcusyn software. Apoptosis was evaluated by Annexin V assay. In vivo efficacy was assessed using an SB-1 (murine CCA cell line) and a syngeneic murine model of CCA treated with vehicle, GemCis, bemcentinib, and the combination of GemCis and bemcentinib. Finally, to investigate the role of AXL Y866 in CCA, we performed BioID to identify and compare the interactomes of AXL WT and AXL Y866F in HuCCT1 cells. Results: In the TCGA cohorts, AXL is significantly expressed in CCA (P < 0.01), and disease-free survival and overall survival in the low AXL expression group are significantly longer than those in the high AXL expression group (P = 0.04, 0.01). In in vitro study, the IC50 value of GemCis was decreased from 685nM to 129nM after AXL knockdown. A synergistic effect was observed with CI = 0.17 and Fa = 0.50 in the combinatorial therapy. The combinatorial therapy caused significantly increased apoptosis compared to GemCis or bemcentinib alone (P < 0.01, P < 0.01). In in vivo study, the combinatorial therapy significantly suppressed the tumor growth compared to GemCis or bemcentinib alone (P = 0.04, 0.01), and the expression levels of Ki67 and the phosphorylation levels of PEAK1 decreased in the combinatorial therapy group compared to other groups in immunostaining examination. In BioID experiments, among the 222 proteins detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was the kinase most affected by AXL Y866. In gene ontology analysis to characterize the interactome of AXL Y866, cell-cell adhesion and focal adhesion-related proteins were enriched. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in the preclinical model. AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA. Citation Format: Shohei Takaichi, Dong-Gi Mun, Jennifer L. Tomlinson, Amro M. Abdelrahman, Danielle M. Carlson, Alaa Abou Daher, Nathan W. Werneburg, Xinyan Wu, Akhilesh Pandey, Gregory J. Gores, Rory L. Smoot. Axl inhibition sensitizes cholangiocarcinoma cells to cytotoxic chemotherapy. [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 4996.

Abstract Background: In phase I/II clinical trials, the tamoxifen metabolite Z-endoxifen (ENDX) demonstrated substantial oral bioavailability and promising antitumor activity in endocrine-refractory estrogen-receptor positive breast cancer (ER+ BC) and other solid tumors, with ENDX plasma concentrations reaching as high as 5 µM, a concentration that far exceeds the ERα-targeting nanomolar concentrations. We previously identified protein kinase C beta 1 (PKCβ1), an oncogenic signaling kinase which regulates cell proliferation and tumorigenic transformation, as a novel ENDX receptor. ENDX-bound PKCβ1 (KD: 100 nM) and inhibited PKCβ1 kinase activity (IC50: 357 nM) in vitro, at concentrations achieved in phase I/II ENDX studies. Given the observations that ENDX induces antitumor activity in breast cancers resistant to endocrine therapies, including AIs, tamoxifen and fulvestrant, we postulated that ENDX may impact additional signaling pathways. Therefore, in this study we performed unbiased mass spectrometry to analyze the effects of ENDX on the phosphoproteome and total proteome. Methods: In estrogen unstimulated aromatase-expressing ER+/human epidermal growth factor 2 receptor negative (HER2-) MCF7AC1 breast cancer cells, the effects of ENDX on the phosphoproteome and total proteome were analyzed using ENDX concentrations achieved in tamoxifen treated patients (0.01 and 0.1 µM) and concentrations observed in the ENDX phase I/II trials (5 µM). NetWorKIN and RoKAI prediction tools were utilized to identify the upstream kinases of ENDX downregulated phosphoproteins. In estrogen unstimulated MCF7AC1 as well as in the ER+/HER2- T47D breast cancer cells, the ability of ENDX, tamoxifen, fulvestrant and PKC kinase inhibitor enzastaurin to block insulin or PKC agonist phorbol 12-myristate 13-acetate (PMA)-stimulated AKTSer473 phosphorylation was analyzed by immunoblot (IB) assay. Additionally, ENDX effects on AKTSer473 phosphorylation in the MCF7AC1 xenograft model in vivo was analyzed by IB assay. Further, the effects of PKCβ1 silencing in MCF7AC1 cells and the effects of AKT inhibitor MK-2206 treatment on growth and AKTSer473 phosphorylation in the MCF7AC1 as well as long-term estrogen deprived T47D (T47D-LTED) cell lines were analyzed by cell proliferation and IB assays. Results: In MCF7AC1 cells unstimulated with estrogen, ENDX at 5 µM but not at 0.01 and 0.1 µM concentrations inhibited growth, and induced apoptosis, suggesting an ERα-independent effect. Further, ENDX at 5 µM displayed three-fold greater effects in downregulating the phosphoproteome compared to the other concentrations, with minimal impact on the total proteome. Protein kinase C beta (PKCβ) and AKT1 were identified as the prevalent upstream kinases for ENDX downregulated protein phosphorylations. Notably, in estrogen unstimulated MCF7AC1 and T47D cells, ENDX at 5 µM attenuated phosphorylation of AKTSer473 and AKT substrates in vitro in the presence of insulin and in vivo. In MCF7AC1 cells, PMA-induced phosphorylation of PKCβ1 as well as AKTSer473 and AKT substrate phosphorylations were blocked by ENDX at 5 µM, with ENDX inducing PKCβ1 protein degradation both in the presence of insulin and PMA. Further, ENDX effects on growth and AKTSer473 phosphorylation were phenocopied by siRNA-mediated PKCβ1 knockdown as well as treatment with the pan-AKT inhibitor, MK-2206. Notably, the potent PKCβ1 kinase inhibitor, enzastaurin, had no effects either on PKCβ1 protein degradation nor on downstream AKT signaling. Conclusion: Taken together, these findings suggest that ENDX may exert anticancer activity via dual effects on blocking ERα as well as by targeting PKCβ1 for degradation, thus suppressing AKT signaling and induction of apoptosis. These preclinical findings will be studied in a planned neoadjuvant trial comparing ENDX with exemestane and ovarian function suppression (EVANGELINE) that will activate in the Fall of 2022. Citation Format: Swaathi Jayaraman, Xinyan Wu, Krishna R. Kalari, Xiaojia Tang, Mary Kuffel, Elizabeth S. Bruinsma, Santosh Renuse, James N. Ingle, Joel Reid, Matthew Schellenberg, John Hawse, Akhilesh Pandey, Matthew P. Goetz. Endoxifen Downregulates AKT Phosphorylation Through Protein Kinase C Beta 1 in ER+/HER2- Breast Cancer. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-04.

Abstract Background: As a key receptor tyrosine kinase (RTK) regulating tumorigenesis, AXL is overexpressed in multiple different types of cancers. AXL can be activated by homodimerization after binding with its ligand, growth arrest specific 6 protein (Gas6) or by heterodimerization with other RTKs. AXL overexpression and/or activation have been reported to promote cancer cell proliferation, migration/invasion and to induce resistance to targeted therapy. However, the AXL downstream signaling network has not yet been fully elucidated. Methods: We employed a CRISPR-CAS9-based knockout technology to create AXL inducible knockout cells in a human triple negative breast cancer (TNBC) cell line, HCC1395. In order to explore the downstream signaling events driven by AXL, we induced AXL knockout for 72 hours and used a 6-plex TMT-labeling-based quantitative proteomic approach to characterize the protein phosphorylation alterations induced by AXL knockout. IMAC and anti-phosphotyrosine antibody (pY1000)-based phosphopeptide enrichment methods were used to comprehensively quantify the phosphoproteome changes induced by AXL knockout. Results: In this study, we identified 38,525 phospho serine/threonine sites and 1,427 phosphotyrosine sites. In response to AXL knockout, 1,581 phospho serine/threonine sites and 40 phosphotyrosine sites showed significant differences between AXL-KO and AXL-WT groups with >1.5-fold change and p<0.05 as the cutoff. Among them, 982 phosphoserine/threonine sites and 30 phosphotyrosine sites were decreased, while 599 phospho serine/threonine sites and 10 phosphotyrosine sites were increased in AXL-KO cells. Signaling pathway enrichment analysis revealed that suppressing AXL expression could inhibit multiple important cancer related signaling pathways, including cell cycle, focal adhesion, MAPK signaling pathway, p53 signaling pathway and mTOR signaling pathway. Conclusion: Quantitative analysis of the phosphoproteome driven by AXL demonstrated that AXL plays a pivotal role in regulating canonical oncogenic signaling but also signaling pathways involved in RNA processing and DNA repair. Our data also suggest that targeting AXL could suppress these oncogenic signaling pathways and have therapeutic potential to improve clinical outcomes for patients with AXL overexpression. Citation Format: Li Wang, Nicole Pearson, Yuning Xiong, Santosh Renuse, Ran Cheng, Jodi M. Carter, Akhilesh Pandey, Xinyan Wu. Quantitative phosphoproteomic analysis of AXL signaling network in breast 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 3913.

Overexpression and amplification of AXL receptor tyrosine kinase (RTK) has been found in several hematologic and solid malignancies. Activation of AXL can enhance tumor-promoting processes such as cancer cell proliferation, migration, invasion and survival. Despite the important role of AXL in cancer development, a deep and quantitative mapping of its temporal dynamic signaling transduction has not yet been reported. Here, we used a TMT labeling-based quantitative proteomics approach to characterize the temporal dynamics of the phosphotyrosine proteome induced by AXL activation. We identified >1100 phosphotyrosine sites and observed a widespread upregulation of tyrosine phosphorylation induced by GAS6 stimulation. We also detected several tyrosine sites whose phosphorylation levels were reduced upon AXL activation. Gene set enrichment-based pathway analysis indicated the activation of several cancer-promoting and cell migration/invasion-related signaling pathways, including RAS, EGFR, focal adhesion, VEGFR and cytoskeletal rearrangement pathways. We also observed a rapid induction of phosphorylation of protein tyrosine phosphatases, including PTPN11 and PTPRA, upon GAS6 stimulation. The novel molecules downstream of AXL identified in this study along with the detailed global quantitative map elucidating the temporal dynamics of AXL activation should not only help understand the oncogenic role of AXL, but also aid in developing therapeutic options to effectively target AXL.