Low Axle Research Articles

Effect of soil compaction and cover crop management on soil physical quality, biomass production and corn and soybean yield

ABSTRACT No-till with cover crop (CC) has been marketed to growers as a sustainable way to increase soil quality and crop yields. Our goal was to better understand the effect of CC management strategies on crop yield and mitigation of soil compaction. The compaction treatments were: four (C4) and eight (C8) wheel passes of a tractor, and without additional compaction (C0). CCs in autumn/winter were Italian ryegrass (IR) hay, IR as CC, and mix of IR and forage radish as CC. In the spring/summer, corn and soybean were cultivated. The reduction of hydraulic conductivity (0–6 cm depth) after first season in the IR hay was 74.9% in C4 and 96.2% in C8 comparted with C0. Overall, harvested CC has limited capacity to respond to compaction, decreasing root growth (root biomass of 2191, 2753, and 3744 kg ha−1, IR hay, IR CC, and mix, respectively). Further, the negative effects of compaction on crop yield were modest, probably because of the low axle loads and contact stresses; high inherent structural stability of Oxisols; and permanent no-till and CC roots which favored soil structure stability. Future works should use higher axle loads and surface stresses and a control treatment without CC.

Abstract 431: FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor

Abstract Metastatic melanoma is one of the most aggressive skin cancers and is associated with poor prognosis. BRAF and MEK inhibitors are used to treat patients with BRAFV600E-mutated advanced melanoma. However, the development of resistances to these treatments compromises therapeutic success. Our lab previously demonstrated that forkhead box D1 (FOXD1) plays a critical role in melanoma migration and invasion. Here, we found that FOXD1 was highly expressed in melanoma cells. Immunohistochemical assessment of 105 samples from patients with metastatic melanoma revealed that high FOXD1 expression in tumors was associated with poor survival and correlated with low MITF, SOX10 and high AXL expression. Upregulation of FOXD1 expression enhanced the resistance of melanoma to vemurafenib (BRAF inhibitor) or combinatorial treatment with vemurafenib and cobimetinib (MEK inhibitor). On the other hand, loss of FOXD1 increased the sensitivity of naïve melanoma cells towards vemurafenib or combinatorial treatment with vemurafenib and cobimetinib. Furthermore, high FOXD1 expression levels were found in BRAF inhibitor (BRAFi)-resistant cells. Downregulation of FOXD1 resulted in a resensitization of BRAFi-resistant cells to vemurafenib. By using microarray analysis, connective tissue growth factor (CTGF) was found to be one of the most downregulated genes in FOXD1 knockdown (KD) cells while its expression was highly increased upon FOXD1 overexpression. Thus, CTGF was identified as a downstream factor of FOXD1. In addition, in vitro expression analysis and evaluation of clinical samples demonstrated that CTGF and FOXD1 expression were positively correlated. By using a CHIP assay and a dual reporter luciferase assay, we discovered that FOXD1 could regulate the expression of CTGF by directly binding to the CTGF promoter. This result was confirmed with RT-PCR and western blot. In addition, we found that the protein level of CTGF was highly increased in BRAFi-resistant cells. Similar to FOXD1 knockdown, the knockdown of CTGF resensitized BRAFi-resistant cells to vemurafenib. FOXD1 KD cells treated with recombinant CTGF protein were less sensitive towards vemurafenib compared to untreated FOXD1 KD cells. Based on these findings, we conclude that the transcription factor FOXD1 could promote dedifferentiation and targeted therapy-resistance in melanoma cells by regulating the expression of CTGF. FOXD1 might be a promising new diagnostical marker and a therapeutic target of targeted therapy resistant melanoma. Citation Format: Qian Sun, Daniel Novak, Laura Hüser, Juliane Poelchen, Huizi Wu, Karol Granados, Aniello Federico Federico, Ke Liu, Tamara Steinfass, Marlene Vierthaler, Viktor Umansky, Jochen Utikal. FOXD1 promotes dedifferentiation and targeted therapy resistance in melanoma by regulating the expression of connective tissue growth factor [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 431.

Effect of AXL on cholangiocarcinoma survival and sensitivity to cytotoxic chemotherapy.

591 Background: Cholangiocarcinoma (CCA) is a lethal disease with limited therapeutic options. We have demonstrated the interaction of Src-family kinase LCK with AXL, a TAM receptor tyrosine kinase (RTK), by phosphoproteomic analysis in CCA ( J Hepatol 2022). AXL is reported to act as a mechanism of acquired drug resistance in solid cancers. However, the role of AXL in CCA remains to be elucidated. Here, we investigated the significance of AXL expression as a potential therapeutic target in CCA. Methods: We first evaluated the expression levels of AXL in CCA and the associations with patient outcome using publicly available data from The Cancer Genome Atlas (TCGA). Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GemCis) therapy as a standard therapy. AXL downregulation was achieved via siRNA approach and the selective inhibitor bemcentinib. We examined 50% inhibitory concentration (IC50) value of HuCCT1, a well characterized CCA cell line, on GemCis therapy with and without AXL knockdown using CellTiter-Glo cell viability assay. Then we assessed the efficacy of the combinatorial therapy of GemCis and bemcentinib utilizing Calcusyn software. Finally, apoptosis was evaluated by Annexin V assay. In vivo efficacy was assessed using a SB-1 (murine CCA cell line) and syngeneic murine model of CCA in C57Bl/6J mice treated with vehicle, GemCis, bemcentinib, and the combination of GemCis and bemcentinib. Results: In the TCGA cohorts, AXL is significantly expressed in CCA (P < 0.01), and disease-free survival and overall survival in low AXL expression group are significantly longer than those in high AXL expression group (P = 0.04, 0.01). In in vitro study, IC50 value of GemCis was decreased from 685nM to 129nM after AXL knockdown. Synergy effect was observed with the value of 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). Conclusions: Targeting AXL sensitizes CCA cell lines and preclinical models to standard of care GemCis combinatorial therapy. Our study suggests that the addition of AXL targeted therapy to cytotoxic chemotherapy can increase the response in CCA patients and is a promising combination.

High levels of AXL expression in untreated EGFR-mutated non-small cell lung cancer negatively impacts the use of osimertinib.

For non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations, the initial therapeutic interventions will have crucial impacts on their clinical outcomes. Drug tolerant factors reportedly have an impact on EGFR-tyrosine kinase inhibitor sensitivity. This prospective study investigated the impacts of drug tolerant-related protein expression in tumors based on the efficacy of osimertinib in the first-setting of EGFR-mutated advanced NSCLC patients. A total of 92 patients with EGFR-mutated advanced or postoperative recurrent NSCLC were analyzed and treated with osimertinib at 14 institutions in Japan. AXL, p53, and programmed death-ligand 1 (PD-L1) expression in patient tumors was determined using immunohistochemistry. The AXL signaling pathway was investigated using a cell line-based assay and AXL-related gene expression in The Cancer Genome Atlas (TCGA) database. High levels of AXL and positive-p53 expression were detected in 26.1% and 53.3% of the pretreatment EGFR-mutated NSCLC tumors, respectively. High AXL expression levels were significantly associated with a shorter progression-free survival compared with low AXL expression levels, irrespective of the EGFR activating mutation status (p= 0.026). Cell line-based assays indicated that the overexpression of AXL protein accelerated PD-L1 expression, which induced insensitivity to osimertinib. In the TCGA database, AXL RNA levels were positively correlated with PD-L1 expression in the lung adenocarcinoma cohort. The results show that high AXL expression levels in tumors impact clinical predictions when using osimertinib to treat EGFR-mutated NSCLC patients. Trial Registration: UMIN000043942.

Cooperative induction of receptor tyrosine kinases contributes to adaptive MAPK drug resistance in melanoma through the PI3K pathway.

Vemurafenib-induced drug resistance in melanoma has been linked to receptor tyrosine kinase (RTK) upregulation. The MITF and SOX10 genes play roles as master regulators of melanocyte and melanoma development. Here, we aimed to explore the complex mechanisms behind the MITF/SOX10-controlled RTK-induced drug resistance in melanoma. To achieve this, we used a number of molecular techniques, including melanoma patient data from TCGA, vemurafenib-resistant melanoma cell lines, and knock-down studies. The melanoma cell lines were classified as proliferative or invasive based upon their MITF/AXL expression activity. We measured the change of expression activity for MITF/SOX10 and their receptor (AXL/ERBB3) and ligand (NRG1/GAS6) targets known to be involved in RTK-induced drug resistance after vemurafenib treatment. We find that melanoma cell lines characterized as proliferative (high MITF low AXL), transform into an invasive (low MITF, high AXL) cell state after vemurafenib resistance, indicating novel feedback loops and advanced compensatory regulation mechanisms between the master regulators, receptors, and ligands involved in vemurafenib-induced resistance. Together, our data disclose fine-tuned mechanisms involved in RTK-facilitated vemurafenib resistance that will be challenging to overcome by using single drug targeting strategies against melanoma.

Abstract LB084: AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601)

Abstract Background: Adenoid cystic carcinoma (ACC) is a rare cancer of salivary gland cancer with a significant unmet clinical need. Conventional systemic therapy has limited efficacy and there are currently no FDA approved drugs for these patients with metastatic disease. Advances in molecular profiling led identification of potential therapeutic targets, and previous studies with proteomic analysis identified AXL as a potential therapeutic target in ACC. AXL, a member of the TAM tyrosine kinase receptor family has been generally associated with poor prognosis, and its overexpression and activation is implicated in conferring resistance to conventional systemic therapies in solid tumors. ADCT-601 is an antibody drug conjugate targeting human AXL carrying novel pyrrolobenzodiazepine dimer cytotoxin as the payload. Methods: The goal of this study is to determine the in vitro and in vivo anti-tumor activity of ADCT-601 in ACC cell lines and patient-derived xenograft (ACCX) models with varying AXL expression determined by Western blot. ADCT-601 treated cell lines in vitro were analyzed by MTT-based cytotoxic assay and immunoblotting, and anti-tumorigenicity was assessed in two ACCX models. Results: ADCT-601 induced a potent dose-dependent cytotoxic effect in ACC cell lines, but its cytotoxic activity was attenuated after AXL knockdown. Biochemical data indicated that ADCT-601 treatment led to DNA damage and induction of apoptotic factors accompanied by phosphorylation of gamma-H2AX. In high AXL expressing ACCX6 model, a single dose of either 0.5 or 1.0 mg/kg ADCT-601 significantly suppressed tumor growth resulting 3/5 partial responders (PRs) at 1mg/kg and 2/5 PRs at 0.5 mg/kg, compared to the vehicle or isotype-ADC control treated mice (0/5 PR and 1/5 PR, respectively). ADCT-601 appears to eliminate tumor, as no evidence of recovery in tumor growth was observed during a 60-day period. In low AXL expressing ACCX9 model, ADCT-601 treatment (at either the 0.5 or 1.0 mg/kg dose) delayed tumor growth compared to isotype-ADC control, but did not lead to durable partial responses. Conclusion: This study demonstrated that ADCT-601 induced a potent and specific in vitro and in-vivo anti-tumor activities in AXL expressing ACC models and suggests further development of ADCT-601 in biomarker driven clinical trials. Citation Format: Joseph O. Humtsoe, Leilani Jones, Shorook Naara, Francesca Zammarchi, Nicole Spardy Burr, Patrick H. van Berkel, Patrick K. Ha, Hyunseok Kang. AXL as a therapeutic target in adenoid cystic carcinoma: preclinical evaluation of AXL targeting antibody-drug conjugate (ADCT-601) [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 LB084.

Abstract 2429: Tumor and stromal AXL expression regulate ovarian cancer metastasis

Abstract Objective: Over 75% of patients with ovarian cancer present with widely metastatic disease. We have previously published that AXL is highly expressed in primary ovarian cancer tumors. However, it is unknown whether AXL expression in metastatic tumors correlates with outcome. Additionally, the role of stromally expressed AXL in metastasis has not been fully evaluated. Thus, the objective of this study is to evaluate the role of tumor and stromal AXL in the key steps of tumor metastasis. Methods: Immunohistochemistry was used to measure AXL expression in a tissue microarray with primary and metastatic high grade ovarian tumors. Attachment assays as well as transwell and spheroid invasion assays were performed on established ovarian cancer cell lines. Mesothelial cell clearance assays utilizing human omentum-cultured mesothelial cells (HPMC) and additional invasion assays utilizing normal omental fibroblasts (NOF) were performed. Results: 214 tumor samples were included from 139 patients: 122 primary and 92 metastatic sites, contributing to 85 matched samples. Most had high-grade serous histology (79%) and stage III-IV disease (79%). High AXL expression in metastatic tumors was associated with decreased overall survival (HRadj 1.8, 95% CI 1.03-3.1), but expression in primary tumors was not (1.1, 0.61-2.0). High vs. low AXL expression in metastases was associated with a decreased platinum-free interval (5.92 vs 15.9 months, P=0.03) and decreased progression-free survival (11.6 vs 18.8 months, P=0.02).Tumor AXL expression correlated with stromal AXL expression (R2=0.50, P<0.001). Human ovarian tumor cells depleted of AXL had decreased attachment (OVCAR3-TPMES, 0.55-fold decrease P<0.001) and invasion in transwell (OVCAR5, 173 vs 262 invasive tumor cells/hpf, P<0.01) and spheroid (ES2, 87% vs 31% area invaded, P<0.0001) invasion assays. Human ovarian cancer cells plated on AXL-deficient HPMCs had less cell clearance than those plated on AXL-expressing HPMCs (ES2, 6.0 vs 3.9 normalized area cleared, P<0.001). Further, human ovarian cancer cells plated on AXL-deficient NOFs had less invasion than tumor cells plated on AXL expressing NOFs (OVCAR5, 173 vs 262 invasive tumor cells/hpf, P<0.01). Conclusions: High AXL expression in metastatic ovarian cancer cells correlates with worse survival and worse response to chemotherapy. Further, AXL expression in both tumor and stromal cells contributes to the steps of ovarian cancer metastasis. Citation Format: Mary Margaret Mullen, Lillian N. van Biljon, Jeanne Quinn, Molly Greenwade, Gregory Opara, Hollie Noia, Ian S. Hagemann, Andrea R. Hagemann, Lindsay M. Kuroki, Carolyn K. McCourt, Premal H. Thaker, Matthew A. Powell, David G. Mutch, Dineo Khabele, Katherine C. Fuh. Tumor and stromal AXL expression regulate ovarian cancer metastasis [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 2429.

Outcomes based on plasma biomarkers in METEOR, a randomized phase 3 trial of cabozantinib vs everolimus in advanced renal cell carcinoma

BackgroundIn the phase 3 METEOR trial, cabozantinib improved progression-free survival (PFS) and overall survival (OS) versus everolimus in patients with advanced RCC after prior antiangiogenic therapy.MethodsIn this exploratory analysis, plasma biomarkers from baseline and week 4 from 621 of 658 randomized patients were analyzed for CA9, HGF, MET, GAS6, AXL, VEGF, VEGFR2, and IL-8. PFS and OS were analyzed by baseline biomarker levels as both dichotomized and continuous variables using univariate and multivariable methods. For on-treatment changes, PFS and OS were analyzed using fold change in biomarker levels at week 4. Biomarkers were considered prognostic if p < 0.05 and predictive if pinteraction < 0.05 for the interaction between treatment and biomarker.ResultsHazard ratios for PFS and OS favored cabozantinib versus everolimus for both low and high baseline levels of all biomarkers (hazard ratios ≤0.78). In univariate analyses, low baseline HGF, AXL, and VEGF were prognostic for improvements in both PFS and OS with cabozantinib, and low HGF was prognostic for improvements in both PFS and OS with everolimus. Low AXL was predictive of relative improvement in PFS for cabozantinib versus everolimus. Results were generally consistent when baseline biomarkers were expressed as continuous variables, although none were predictive of benefit with treatment. In multivariable analysis, low baseline HGF was independently prognostic for improved PFS for both cabozantinib and everolimus; low HGF, GAS6, and VEGF were independently prognostic for improved OS with cabozantinib. No biomarkers were independently prognostic for OS with everolimus. On-treatment increases in some biomarkers appeared prognostic for PFS or OS with cabozantinib in univariate analyses; however, none were independently prognostic in multivariable analysis.ConclusionsPFS and OS were improved with cabozantinib versus everolimus at high and low baseline levels of all biomarkers. Low baseline HGF was consistently identified as a prognostic biomarker for improved PFS or OS with cabozantinib or everolimus, supporting further prospective evaluation of the prognostic significance of HGF in advanced RCC.Trial registrationClinicalTrials.gov NCT01865747 (registered on 05/31/2013).

Abstract 1075: AXL is a potential druggable target in trastuzumab resistance in HER2+ breast cancer patients

Abstract Introduction HER2+ correlates with aggressive phenotype and poor prognosis. The use of several antiHER2 drugs has dramatically improved prognosis and survival of HER2+ breast cancer (BC) patients. However, ≈20% of these patients will present resistance to antiHER2 therapies and relapse. The purpose of this study is to explore the role of AXL in antiHER2 treatment resistance and its potential as a druggable biomarker in HER2+ BC. Methods We generated 2 trastuzumab (T) resistant models from a HER2+ BC patient. The first one was a PDX derived resistant cell line established by chronic treatment for 6 months with T in vitro (TR1 and TR2). The second resistant model (TR4) was established by chronic treatment with T for 4 months and two serial passages in mice. The prognostic value of AXL was evaluated in primary tumor cohort of HER2+ BC patients treated by standard guidelines (n=51). AXL was also explored in a cohort from PAMELA trial, a neoadjuvant phase II study. Patients were given lapatinib and T for 18 weeks (Luminal-HER2+ patients were additionally given hormonotherapy). Tumor samples were collected at baseline, day 14 and surgery (n= 96). Results PDX resistant cell lines (TR1 and TR2) and resistant tumors (TR4) exhibit upregulation of AXL in comparison to the parental cells (PDX118). In vitro, the combination of an AXL inhibitor (TP-0903) + T resensitized resistant cells to T. In 3D organoids models from TR4, the combination decreased cell number in organoids more than both single agents. In TR4 in vivo model, TP-0903 + T completely abrogate tumor growth for more than 2 months.To validate our preclinical findings, we analyzed the level of AXL in our retrospectively cohort of patients. AXL level was significantly upregulated in patients who relapsed (p&amp;lt;0.0001). Patients with high levels of AXL had significantly shorter disease-free survival (DFS) and overall survival (OS) (log-rank p&amp;lt;0.0001; HR=15.78; 95% CI 3.89-19.80 for DFS; log-rank p=0.013; HR= 5.43; 95% CI 1.35-13.07 for OS). The median DFS and OS were 36 and 77 months in patients with high AXL level, whereas patients with low AXL level presented 92.5 and 92.5 months.To identify early molecular changes induced by dual HER2 blockade in patients with HER2+ disease, AXL was analyzed in PAMELA trial cohort. A significant increase of AXL was detected at day 14 compared to baseline (p=5.8e-20). This effect was observed in both luminal and non-luminal HER2+ BC patients and across all PAM50 subtypes. This rapid response suggests the implication of AXL as a mechanism of antiHER2 resistance. Levels of AXL decreased as long as treatment was on hold at surgery compared to day 14 (p=6.04e-14). Conclusion AXL level was correlated with poor outcome of HER2+ BC patients. On our preclinical models, we showed that combination of TP-0903 and T abrogate tumor growth in acquired resistant models to T in PDXs. We postulate AXL as a promising new therapeutic strategy to overcome resistance to antiHER2 therapies Citation Format: Anna Adam-Artigues, Raimundo Cervera, Enrique Javier Arenas, Fara Brasó-Maristany, Alex Martinez-Sabadell, Eduardo Tormo, Cristina Hernando, Maria Teresa Martinez, Soraya Simon, Jesús Poveda, Octavio Burgués, Ana Rovira, Federico Rojo, Joan Albanell, Begoña Bermejo, Ana Lluch, Pilar Eroles, Aleix Prat, Joaquin Arribas, Juan Miguel Cejalvo. AXL is a potential druggable target in trastuzumab resistance in HER2+ breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1075.

Permeable pavements for storm water control incorporating nano clay

Stormwater affects the economic situation of the environment, changes the land use, which also affects the ecosystem. Consequently, this affects the pavement condition of the road and lead to both structural and functional pavement defects. In a bid to proffer solution to the harmful effects of stormwater and reduce the cost of pavement construction in track with sustainability, this research utilized nano clay in the development of permeable concrete. The nano clay was used as a partial replacement for cement. It was dehydroxylated at 720°C; the characterization and particle geometry was done using the XRF equipment. The de-hydroxylated nano clay was used as a partial replacement for cement at 5, 10, 15, 20 and 25%. Correspondingly, six samples of concrete were developed. The mechanical properties (compressive and flexural strength) of the permeable concrete was assessed at 3, 7, 14, 21 and 28 days. Additionally, the porosity of the concrete was determined using Archimedes principle. The result of the research showed that using nano clay replacement at 15% gave a flexural strength of 4.01MPa. This is lower than the required specification of 4.27Mpa—4.5MPa for pavement construction at 28 days and 4.5Mpa and above for airfield pavement. The porosity test using Archimedes principle showed a satisfactory result. This proves the efficiency of the concrete for stormwater control. Therefore, this concrete is recommended for use in the design and construction of low axle or low trafficked road for stormwater control and aquifer recharge based on the flexural strength.

Exosome-transmitted linc00852 associated with receptor tyrosine kinase AXL dysregulates the proliferation and invasion of osteosarcoma.

BackgroundReceptor tyrosine kinase AXL has been found to be highly expressed in osteosarcoma and positively associated with poor prognosis. There are tumor groups with high or low AXL expression, which had different capabilities of invading vessels and forming distal metastases. Exosome‐transmitted lncRNA may be transferred intercellularly to promote tumor cells’ proliferation and invasion.MethodsExosomes were detected by electron microscopy, particle size analysis, and western blotting. High‐throughput sequencing helped to find the highest differentially expressed lncRNA in AXL‐associated exosomes. Clone formation, wound healing, transwell assay, and xenograft model in nude mice were performed to evaluate cells’ proliferation, migration, and invasion in vitro and in vivo. Lentiviral transfection was used to up‐ or down‐regulate the lncRNA levels in cell lines. Luciferase reporter assay and RNA FISH etchelped to indicate the molecular mechanisms. The results in the cell lines were proved in the osteosarcoma tissues with clinical analysis.ResultsThe exosomes derived from donor cells with high AXL expression could promote the proliferation and invasion and upregulate AXL expression of the receiver cells with low AXL. Linc00852 was the highest differentially expressed lncRNA in AXL‐associated exosomes and was also regulated by AXL expression. Although the mechanisms of linc00852 in nucleus were unrevealed, it could upregulate AXL expression partly by competitively binding to miR‐7‐5p. The AXL‐exosome‐linc00852‐AXL positive feedback loop might exist between the donor cells and the receiver cells. Clinically, linc00852 was significantly highly expressed in osteosarcoma tissues and positively associated with tumor volumes and metastases, which was also obviously related with AXL mRNA expression.ConclusionAXL‐associated exosomal linc00852 up‐regulated the proliferation, migration, and invasion of osteosarcoma cells, which would be considered as a new tumor biomarker and a special therapeutic target for osteosarcoma.

Hematopoietic progenitor kinase 1 down-regulates the oncogenic receptor tyrosine kinase AXL in pancreatic cancer

The oncogenic receptor tyrosine kinase AXL is overexpressed in cancer and plays an important role in carcinomas of multiple organs. However, the mechanisms of AXL overexpression in cancer remain unclear. In this study, using HEK293T, Panc-1, and Panc-28 cells and samples of human pancreatic intraepithelial neoplasia (PanIN), along with several biochemical approaches and immunofluorescence microscopy analyses, we sought to investigate the mechanisms that regulate AXL over-expression in pancreatic ductal adenocarcinoma (PDAC). We found that AXL interacts with hematopoietic progenitor kinase 1 (HPK1) and demonstrate that HPK1 down-regulates AXL and decreases its half-life. The HPK1-mediated AXL degradation was inhibited by the endocytic pathway inhibitors leupeptin, bafilomycin A1, and monensin. HPK1 accelerated the movement of AXL from the plasma membrane to endosomes in pancreatic cancer cells treated with the AXL ligand growth arrest-specific 6 (GAS6). Moreover, HPK1 increased the binding of AXL to the Cbl proto-oncogene (c-Cbl); promoted AXL ubiquitination; decreased AXL-mediated signaling, including phospho-AKT and phospho-ERK signaling; and decreased the invasion capability of PDAC cells. Importantly, we show that AXL expression inversely correlates with HPK1 expression in human PanINs and that patients whose tumors have low HPK1 and high AXL expression levels have shorter survival than those with low AXL or high HPK1 expression (p < 0.001). Our results suggest that HPK1 is a tumor suppressor that targets AXL for degradation via the endocytic pathway. HPK1 loss of function may contribute to AXL overexpression and thereby enhance AXL-dependent downstream signaling and tumor invasion in PDAC.

Receptor Tyrosine Kinase AXL: A Potential Strategy to Counter Immune Suppression and Dormancy in Multiple Myeloma

Introduction The AXL receptor tyrosine kinase (AXL) has emerged as a promising therapeutic target for cancer therapy. Recent studies revealed a crucial role of AXL signaling in proliferation, survival, dormancy and therapy resistance in different cancers including lung cancer, hepatocellular cancer and AML. In this study, we aimed to investigate the role of AXL in Multiple Myeloma (MM), focusing on myeloma cell dormancy and AXL expression in different cellular components of the bone marrow microenvironment. Material &amp; Methods To investigate dormancy, we used the syngeneic murine 5TGM1 MM model. 5TGM1-GFP+cells were DiD-labeled and injected intravenously in naïve C57BL/KaLwRij mice. At end-stage, GFP+DiD+('dormant', non-proliferating) and GFP+DiD-('proliferating') MM cells were analyzed by flow cytometry for AXL expression. In addition, AXL expression was also analyzed in CD11b+ myeloid cells and in in vitrogenerated macrophages from the 5TMM model. The effects of AXL inhibition by R428 (BGB324|Bemcentinib, Sigma-Aldrich), a highly potent and AXL-specific small molecular inhibitor, on viability and induced apoptosis of MM cells was determined by Cell Titer Glo and AnnexinV/7AAD staining respectively. AXL expression in human myeloma cell lines (HMCL) (JJN3, U266 and LP-1) and murine 5TGM1 cells was analyzed by qRT-PCR and cytospin stainings. Patient cohorts (TT2/TT3) were used to correlate AXL expression and overall survival. Plasma of healthy donors and MM patients was analyzed by ELISA (R&amp;D). Results Using the in vivo5TGM1 dormancy model, we demonstrated an increased expression of AXL (4x higher) in dormant MM cells compared to proliferating MM cells (n=3, p&lt;0,05). Myeloma cell lines (JJN3, U266, 5TGM1) had a very low AXL expression, however, treatment with melphalan induced a more than twofold increase in AXL expression (n=3, p&lt;0.05). The combination of melphalan and R428 significantly increased apoptosis of JJN3 (&gt;10%), U266 (&gt;20%) and LP-1 (&gt;10%) cells compared to single agent therapy (n=6) (p&lt;0.01). Using patient cohorts, we observed that AXL expression correlated with a good overall survival (p=0.006). In addition, plasma samples of patients (n=31) showed a decreased expression of AXL compared to samples of healthy controls (n=9) (p&lt;0.001). This confirms our hypothesis that AXL is associated with dormancy and therefore correlates with a better overall survival. In a second part, we investigated AXL expression in 5TMM-derived myeloid cells and macrophages (n=3). We observed a high expression of AXL in myeloid derived suppressor cells and tumor associated macrophages compared to myeloma cells. In addition, we observed that myeloid cells were much more sensitive to R428 compared to MM cells (n=5, p&gt;0.01). Conclusion We observed that AXL is highly expressed in dormant MM cells and environmental myeloid cells. Despite its association with a good prognosis in MM, AXL serves as an interesting target to eradicate dormant myeloma cells as AXL inhibitors affect viability and induce apoptosis of myeloma cells, especially in combination with melphalan. Therefore, AXL can be considered as a new therapeutic strategy, to target both the immunosuppressive myeloid cells and the residual cancer cells in MM patients. Disclosures No relevant conflicts of interest to declare.

Load equivalency factors for off-road trucks

Abstract This article addresses an analysis of the Load Equivalency Factor (LEF) for off-road trucks. A new LEF curve for single axles with dual wheels covering trucks from 6.0 to 151.42 tons per axle is proposed, converting various magnitudes of damage from wheel loads to damage caused by the standard axle load of 8.2 tons. Then, this damage ratio was raised to the exponent proposed by Pereira (1992), and LEF for the considered loads was obtained. In the determination of LEF from the structural response of the subgrade, it was confirmed that LEF values did not suffer significant variations with the various parameters adopted, encompassing variations in the axle loads between 6 and 151.42 tons, tire pressures of 80.0, 100.0 and 120.0 psi, as well as five different pavement structures. In the study, LEF remained stable even in pavement structures with low and high axle capacities. In order to validate the results, the resulting factors were then compared with those proposed by DNIT (2006), displaying a coefficient of determination of 0.99. The conclusion is that pavements for off-roads trucks can be designed using the procedure recommended by DNIT (2006) for flexible pavement, without extrapolation of the respective LEF curve.

Long‐term effects of vehicular passages on soil carbon sequestration and carbon dioxide emission in a no‐till corn‐soybean rotation on a Crosby silt loam in Central Ohio, USA

AbstractResearch information from a systematic planned study on the effects of vehicular passages and axle load on soil carbon dioxide (CO2) fluxes and soil carbon (C) sequestration under long‐term NT farming is scanty. Therefore, the present study was conducted on an on‐going 20‐year experiment to assess the impacts of variable vehicular passages of a low axle load on soil CO2 emission and soil C sequestration from a no‐till (NT) managed corn (Zea mays L.)–soybean (Glycine max Linneo) rotation in comparison with that a soil under woodlots (soils under natural wooded plantation). The experimental treatment consisted of an empty wagon [0 Mg load for compaction (C‐0; control)] compared with 2 (C‐2) and 4 (C‐4) passages of 2.5 Mg water wagon axle load, applied to the entire plot every year during April/May for 20 consecutive years. Soil samples were obtained in November 2016 to determine the effects of various vehicular passages on C and nitrogen (N) contents and CO2 emissions. Soil CO2 fluxes were measured from November 16, 2016, to May 30, 2017, on the bi‐weekly (November to December and April to May) and monthly (January to March) basis by using high‐density polyvinyl chloride static gas chambers. The soil CO2 fluxes ranged from –1.05 to 9.03 g CO2 m−2 d−1. The lowest soil CO2 fluxes were observed in December coinciding with the minimum soil temperature. In general, daily soil CO2 fluxes were higher under C‐0 than those under other treatments. Vehicular traffic and axle load reduced the cumulative emission of CO2 by 22.6 and 29.8% under C‐2 and C‐4, respectively, compared with that under C‐0 (6.09 Mg ha−1). Soil and air temperatures had a significant positive correlation with the diurnal fluxes of soil CO2 in all the treatments except that under C‐4. Electrical conductivity, soil C and N contents and pools did not differ significantly among the treatments. Further, 2 to 4 passages of vehicles with 2.5 Mg of axle load decreased the soil CO2 emission on Crosby silt loam under NT as compared to that under the control. Therefore, continuous cultivation of row crops with moderate trafficking under NT and residue retention is recommended, and it also reduces the potential of soil CO2 emission while improving the soil organic C pools of well‐drained soils of Central Ohio.

Afatinib Is a New Therapeutic Approach in Chordoma with a Unique Ability to Target EGFR and Brachyury.

Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRβ, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRβ inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603-13. ©2017 AACR.

Highlights of This Issue

A novel DNA alkylating payload of antibody-drug conjugates (ADCs) was designed and evaluated in preclinical cancer models by Millers and colleagues. Changing the mechanism of action of the cytotoxic agent from a crosslinker to a DNA alkylator, along with linker optimization, provided ADCs with improved bystander killing and greater in vivo activity. ADCs with the DNA alkylator also displayed better in vivo tolerability than those with a DNA crosslinker, and thus, a higher in vivo therapeutic index was achieved. These findings represent a significant advancement in ADC technology designed to ultimately offer cancer patients a safer, yet highly active, therapeutic option.Recent advances in cancer genomics and transcriptomic analysis of ovarian cancer suggest dysregulation of many critical genes regulating ovarian cancer cell growth. Among them, histone methyl transferase, EZH2, acts as a global transcriptional repressor and downregulates the expression of many tumor suppressor genes. Multiple specific inhibitors of EZH2 have been identified and are currently in clinical trials. EZH2 expression has been shown to induce platinum resistance in ovarian cancer, and recent studies have suggested that EZH2 inhibition may be a valuable therapeutic approach. This review article summarizes the current literature pertaining to EZH2 in ovarian cancer.Chordomas are rare bone tumors marked by an unfavorable prognosis in most cases, with no efficacious therapy beyond surgery. Magnaghi and colleagues identified afatinib as the only EGFR inhibitor with activity across a panel of chordoma cell lines. The treatment of afatinib also led to tumor regression in different chordoma models in vivo. Further mechanistic studies found that afatinib could promote the degradation of EGFR and brachyury proteins, and the sensitivity of chordoma cell lines to afatinib is correlated with high EGFR phosphorylation and low AXL and STK33 expression. These data provided the preclinical rationale for the upcoming European Phase II clinical study with afatinib in chordoma.Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. Bhakta and colleagues identified and characterized GFRA1, a luminal A (hormone receptor-positive) breast cancer target for its use as an antibody-drug conjugate (ADC) therapeutic. A humanized anti-GFRA1 ADC demonstrated specific in vitro and in vivo killing of GFRA1 expressing cells and displayed ideal therapeutic features, including a target safety profile and pharmacokinetic properties that would enable its progress towards further development into the clinic. GFRA1 ADC has potential utility as a targeted therapy for many-relapsed luminal A breast cancer patients.

Case study of effective current estimation in main circuit of tram vehicle

Currently the trams are experiencing the renaissance and more and more Polish cities decide to renew the fleet. New vehicles are usually characterized by high transport capacity, low floor, climate comfort, crash resistance, low energy consumption and low axle loads. Such requirements can only be achieved through optimization in many areas of the vehicle's construction. One of such tasks is the optimal selection of components of the main circuit installation and traction needs. The paper is oriented on the issue of engineering modelling of the effective values of currents flowing in the main circuit and the tramway traction needs circuits. Due to the non-periodic nature of the vehicle traffic loads in urban traffic, the optimal selection of the performance parameters helps in better selection of conductor cross sections, filters and safeguards. A better selection of these components means a reduction of its weight, which is extremely important for meet basic requirements of modern tram vehicles. The paper is intended to gather the experience of authors in this field of knowledge and compares several modelling strategies that the authors use during designing tram vehicles. The elaboration summarizes the results obtained using different modelling strategies, and shows correlation between effective value and selection of components.

ISY1-1 - Innate resistance in EGFR mutant non-small-cell lung cancer patients by co-activation of receptor tyrosine kinases(RTKs)

The MET ligand HGF in stromal cells provides an alternative signaling mechanism for EGFR by inducing inter-receptor cross talk with EphA2, CDCP1 or AXL. Src-homology 2 domain-containing phosphatase 2 (SHP2) also plays a role in RTK signaling and is required for MAPK activation. We studied the expression levels of stromal HGF and tumor RTKs: AXL, CDCP1, MET, and EphA2, as well as SHP2, and clinical outcome in baseline samples of 64 EGFR-mutant NSCLC patients (pts) treated with first-line EGFR TKI. Paraffin-embedded samples and the discrimination between tumor and stromal cells were performed by a pathologist. mRNA expression was analyzed by qRT-PCR. AXL and CDCP1 were among the genes that most significantly influenced treatment outcome. Median progression-free survival (PFS) was 14 months (mo) and 23.4 mo for pts with high and low AXL mRNA, respectively (p = 0.03), (HR: 2.05, p = 0.03). Median PFS was 9 mo and 20.2 mo for pts with high and low CDCP1 mRNA, respectively (p = 0.01), (HR: 2.1, p = 0.02). There were no significant differences in PFS according to levels of EPHA2 and MET in the tumor, and HGF in the stroma. Median PFS was 11.4 mo and 24 mo for pts with high and low SHP2 mRNA, respectively (p = 0.09) (HR: 2.4, p = 0.01). Median overall survival (OS) was 19.1 mo and 40.7 mo for pts with high and low AXL mRNA, respectively (p = 0.009) (HR: 2.9, p = 0.001). Median OS was 19.1 mo and 34.9 mo for pts with high and low CDCP1 mRNA, respectively (p = 0.03) (HR: 2.2, p = 0.03). In addition, differences in OS were noted according to levels of EPHA2, MET and SHP2. AXL and CDCP1 are adverse predictive markers of PFS and OS. It has been reported that overexpression of several RTKs can substitute EGFR signaling in EGFR mutant NSCLC pts. The findings show the need to scrutinize RTK levels in EGFR mutant NSCLC patients and, henceforth, the importance of tailored combinatory therapy with AXL inhibitors, or with Src inhibitors, in those pts with elevated CDCP1 (CDCP1 triggers SFK activation).

Numerical investigation of the effects of ITD length on low pressure nozzle

The advantage of high efficiency, low SFC (Specific Fuel Consumption), ultra-high bypass ratio turbofan engine attracts more and more attention in modern commercial engine. The intermediate turbine duct (ITD), which connects high pressure turbine (HPT) with low pressure turbine (LPT), has a critical impact on the overall performance of turbine by guiding flow coming from HPT to LPT without too much loss. Therefore, it becomes more and more urgent to master the technique of designing aggressive, even super-aggressive ITD. Much more concerns have been concentrated on the duct. However, in order to further improve turbine, LPT nozzle is arranged into ITD to shorten low pressure axle. With such design concept, it is obvious that LPT nozzle flow field is easily influenced by upstream duct structure, but receives much less interests on the contrary. In this paper, numerical method is used to investigate the effects of length of ITD with upstream swirl blades on LPT nozzle. Nine models with the same swirl and nozzle blades, while the length of ITD is the only parameter to be changed, will be discussed. Finally, several conclusions and advices for designers are summarized. After changing axial length of ducts, inlet and outlet flow field of nozzle differs, correspondingly. On the other hand, the shearing stress on nozzle blade (suction and pressure) surface presents individual feature under various inlet flow. In addition to that, “Clocking-like effect” is described in this paper, which will contribute much to the pressure loss and should be paid enough attention.