scholarly journals OR12-02 When the Glucocorticoid Receptor Meets the Mineralocorticoid Receptor in the Nucleus of Human Cells

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Maria G Petrillo ◽  
Christine M Jewell ◽  
Robert H Oakley ◽  
John A Cidlowski
Keyword(s):  
Glucocorticoid Receptor ◽  
Cell Lines ◽  
Mineralocorticoid Receptor ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Proximity Ligation Assay ◽  
Osteosarcoma Cell ◽  
Proximity Ligation ◽  
Gene Response ◽  
Response To Stress

Abstract Adrenal corticosteroids, such as glucocorticoids and mineralocorticoids, are indispensable for mediating response to stress, development, limiting inflammation, and maintaining energy and fluid homeostasis. These hormones exert their actions via binding to two closely related nuclear receptors, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR). The GR has low affinity for corticosteroids, but is expressed in nearly every cell. In contrast, the MR shows a higher affinity for corticosteroids and its expression is largely confined to those tissues where electrolyte exchange and fluid balance are required. GR and MR act as ligand-activated transcription factors which, following interaction with co-regulators and DNA responsive elements, either promote or repress gene transcription. The affinity for the same ligands, structural homology, and binding to the same DNA regions suggest GR and MR can compensate for each other’s actions. Yet, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate this interplay, we developed U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (GRMR). Immunofluorescence analysis showed that treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear traslocation of GR and MR. Conversely, treatment with 1 nM of aldosterone (Aldo) promoted nuclear translocation of the MR only. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR dimers were detected in the nucleus of GRMR cells. Surprisingly, nuclear GR-MR dimers were also detected in the presence of Aldo, suggesting that it is necessary to activate at least one receptor to induce nuclear traslocation of the heterocomplex. To decipher the functional contribution of GR-MR dimers in the transcriptional response of GR to Dex and MR to Aldo, we performed RNA-seq in GR, MR, and GRMR cells treated with 1 nM of Dex or Aldo. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a blunted Dex-mediated gene response which affected only 1608 genes, suggesting a functional antagonism of MR. Aldo-activated MR regulated the transcription of 1660 genes. However, co-expression of GR expanded the Aldo-mediated gene response to 3150 genes. Strikingly, 74% of these genes were also regulated by Dex via GR, suggesting that GR-MR dimers in the presence of aldosterone are able to mimic the glucocorticod transcriptional response. Our data suggest that the role of distinct GR and MR homo- and hetero-dimers is relevant for regulating gene expression. Dissecting the mechanism and investigating the cross-talk between GR and MR may be useful to understanding these two receptors in heath and disease.

scholarly journals One Hormone for Two Receptors: Exploring Glucocorticoid Actions Mediated by the Glucocorticoid and Mineralocorticoid Receptors

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A812-A813
Author(s):  
Maria G Petrillo ◽  
Christine Jewell ◽  
Carl D Bortner ◽  
Robert H Oakley ◽  
John A Cidlowski
Keyword(s):  
Transcription Factor ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Structural Similarity ◽  
Whole Body ◽  
Proximity Ligation Assay ◽  
Osteosarcoma Cell ◽  
Glucocorticoid Treatment

Abstract Glucocorticoids are indispensable for mediating the response to stress, energy demands, development, and limiting inflammation. Once in the cell, these hormones exert their actions by activating nuclear receptors, transcription factors that regulate gene expression. The glucocorticoid receptor (GR) is the transcription factor that predominantly mediates both physiological and pharmacological glucocorticoid effects. Yet glucocorticoids can also bind and activate the mineralocorticoid receptor (MR), a transcription factor known to bind aldosterone thus maintaining whole-body fluid homeostasis. Phylogenetically, GR and MR are closely related and share a remarkable structural similarity. Indeed, the DNA-binding domain of MR is 96% identical to that of GR; thus MR is recruited to many of the same DNA response elements that bind GR. Moreover, GR has a low affinity for glucocorticoids but is expressed in nearly every cell, whereas MR shows a higher affinity for glucocorticoids although knowledge of MR’s expression levels is somewhat limited. These characteristics suggest that, while GR and MR can compensate for each other’s actions in many tissues, there are specific glucocorticoid and mineralocorticoid-mediated responses indicating GR-MR functional diversity. To investigate the similarities and differences between GR and MR signaling in the presence of glucocorticoid hormones, we generated U-2 OS (human osteosarcoma) cell lines stably expressing GR, MR, and both GR and MR (MRGR). Immunofluorescence analysis showed that the treatment of these cell lines with 1 nM of the synthetic glucocorticoid dexamethasone (Dex) induced nuclear translocation of both GR and MR. Moreover, Proximity Ligation Assay revealed that, in the absence of ligand, GR associated with MR in the cytoplasm and, upon 1 nM Dex exposure, GR-MR complexes were detected in the nucleus of MRGR cells. To decipher the functional contribution of GR-MR complexes in the transcriptional response to Dex, we performed RNA-seq in GR, MR, and MRGR cells treated with 1 nM of Dex. Transcriptome analysis revealed that Dex-activated GR regulated the transcription of 6180 genes. Co-expression of MR resulted in a greatly blunted Dex-mediated gene response which reduced the glucocorticoid-dependent transcriptome size by 75%. This phenomenon was also observed using a higher concentration of Dex. Indeed, 40% of genes commonly regulated by Dex in GR and MRGR cells showed a reduced magnitude of regulation when MR is co-expressed. These results suggest a functional antagonism between GR and MR in which MR inhibits GR function. Understanding the molecular mechanisms governing the cross-talk between GR and MR is crucial for the development of new therapies that address the adverse effects of glucocorticoid treatment as well as for the discovery of novel glucocorticoid-based therapeutics with minimal side effects.

scholarly journals PGRMC1 localization and putative function in the nucleolus of bovine granulosa cells and oocytes

Reproduction ◽  
2018 ◽  
Vol 155 (3) ◽  
pp. 273-282 ◽  
Author(s):  
Laura Terzaghi ◽  
Alberto Maria Luciano ◽  
Priscila C Dall’Acqua ◽  
Silvia C Modina ◽  
John J Peluso ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Granulosa Cells ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Multifunctional Protein ◽  
Proximity Ligation ◽  
Response To Stress ◽  
Interfering Rna ◽  
Cellular Stressors

Progesterone receptor membrane component-1 (PGRMC1) is a highly conserved multifunctional protein that is found in numerous systems, including reproductive system. Interestingly, PGRMC1 is expressed at several intracellular locations, including the nucleolus. The aim of this study is to investigate the functional relationship between PGRMC1 and nucleolus. Immunofluorescence experiments confirmed PGRMC1’s nucleolar localization in cultured bovine granulosa cells (bGC) and oocytes. Additional experiments conducted on bGC revealed that PGRMC1 co-localizes with nucleolin (NCL), a major nucleolar protein. Furthermore, small interfering RNA (RNAi)-mediated gene silencing experiments showed that when PGRMC1 expression was depleted, NCL translocated from the nucleolus to the nucleoplasm. Similarly, oxidative stress induced by hydrogen peroxide (H2O2) treatment, reduced PGRMC1 immunofluorescent signal in the nucleolus and increased NCL nucleoplasmic signal, when compared to non-treated cells. Although PGRMC1 influenced NCL localization, a direct interaction between these two proteins was not detected using in situ proximity ligation assay. This suggests the involvement of additional molecules in mediating the co-localization of PGRMC1 and nucleolin. Since nucleolin translocates into the nucleoplasm in response to various cellular stressors, PGRMC1’s ability to regulate its localization within the nucleolus is likely an important component of mechanism by which cells response to stress. This concept is consistent with PGRMC1’s well-described ability to promote ovarian cell survival and provides a rationale for future studies on PGRMC1, NCL and the molecular mechanism by which these two proteins protect against the adverse effect of cellular stressors, including oxidative stress.

scholarly journals Activation of PAR2 by tissue factor induces the release of the PTEN from MAGI proteins and regulates PTEN and Akt activities

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Mohammad A. Mohammad ◽  
John Greenman ◽  
Anthony Maraveyas ◽  
Camille Ettelaie
Keyword(s):  
Tissue Factor ◽  
Phosphatase Activity ◽  
Cell Lines ◽  
Prolonged Exposure ◽  
Term Treatment ◽  
Proximity Ligation Assay ◽  
Continuous Exposure ◽  
Short Term Treatment ◽  
Cellular Survival ◽  
Proximity Ligation

AbstractTissue factor (TF) signalling has been associated with alterations in Akt activity influencing cellular survival and proliferation. TF is also shown to induce signalling through activation of the protease activated receptor (PAR)2. Seven cell lines were exposed to recombinant-TF (rec-TF), or activated using a PAR2-agonist peptide and the phosphorylation state of PTEN, and the activities of PTEN and Akt measured. Furthermore, by measuring the association of PTEN with MAGI proteins a mechanism for the induction of signalling by TF was proposed. Short term treatment of cells resulted in de-phosphorylation of PTEN, increased lipid-phosphatase activity and reduced Akt kinase activity in most of the cell lines examined. In contrast, continuous exposure to rec-TF up to 14 days, resulted in lower PTEN antigen levels, enhanced Akt activity and increased rate of cell proliferation. To explore the mechanism of activation of PTEN by TF, the association of "membrane-associated guanylate kinase-with inverted configuration" (MAGI)1–3 proteins with PTEN was assessed using the proximity ligation assay and by co-immunoprecipitation. The interaction of PTEN with all three MAGI proteins was transiently reduced following PAR2 activation and explains the changes in PTEN activity. Our data is first to show that PAR2 activation directly, or through exposure of cells to TF releases PTEN from MAGI proteins and is concurrent with increases in PTEN phosphatase activity. However, prolonged exposure to TF results in the reduction in PTEN antigen with concurrent increase in Akt activity which may explain the aberrant cell survival, proliferation and invasion associated with TF during chronic diseases.

scholarly journals IKKα plays a major role in canonical NF-kB signalling in colorectal cells

2022 ◽  
Author(s):  
Jack A Prescott ◽  
Kathryn Balmanno ◽  
Jennifer P Mitchell ◽  
Hanneke Okkenhaug ◽  
Simon J Cook
Keyword(s):  
Cell Lines ◽  
Nuclear Translocation ◽  
Transcriptional Response ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Knock Out ◽  
Iκb Kinase ◽  
Sw620 Cells ◽  
Hct116 Cells

Inhibitor of kappa B (IκB) kinase β (IKKβ) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKα being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKα and IKKβ in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKβ inhibitors. IKKα and IKKβ were redundant for IκBα phosphorylation and turnover since loss of IKKα or IKKβ alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKα was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFα and IL-1α. In these cells IKKβ was far less efficient at compensating for the loss of IKKα than IKKα was able to compensate for the loss of IKKβ. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKβ inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKα loss strongly inhibited TNFα-dependent p65 nuclear translocation, IKKα and IKKβ contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKα in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKβ to date.

scholarly journals Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

Cells ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 1631
Author(s):  
Cole P. Frisbie ◽  
Alexander Y. Lushnikov ◽  
Alexey V. Krasnoslobodtsev ◽  
Jean-Jack M. Riethoven ◽  
Jennifer L. Clarke ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Brefeldin A ◽  
Direct Interaction ◽  
Complete Recovery ◽  
Proximity Ligation Assay ◽  
Conformational Structure ◽  
Force Microscopy ◽  
Proximity Ligation ◽  
Golgi Membranes ◽  
Response To Stress

Background: The Golgi apparatus undergoes disorganization in response to stress, but it is able to restore compact and perinuclear structure under recovery. This self-organization mechanism is significant for cellular homeostasis, but remains mostly elusive, as does the role of giantin, the largest Golgi matrix dimeric protein. Methods: In HeLa and different prostate cancer cells, we used the model of cellular stress induced by Brefeldin A (BFA). The conformational structure of giantin was assessed by proximity ligation assay and atomic force microscopy. The post-BFA distribution of Golgi resident enzymes was examined by 3D SIM high-resolution microscopy. Results: We detected that giantin is rather flexible than an extended coiled-coil dimer and BFA-induced Golgi disassembly was associated with giantin monomerization. A fusion of the nascent Golgi membranes after BFA washout is forced by giantin re-dimerization via disulfide bond in its luminal domain and assisted by Rab6a GTPase. GM130-GRASP65-dependent enzymes are able to reach the nascent Golgi membranes, while giantin-sensitive enzymes appeared at the Golgi after its complete recovery via direct interaction of their cytoplasmic tail with N-terminus of giantin. Conclusion: Post-stress recovery of Golgi is conducted by giantin dimer and Golgi proteins refill membranes according to their docking affiliation rather than their intra-Golgi location.

scholarly journals Proximity Ligation Assay as a Tool for Antibody Validation in Human Tissues

2020 ◽  
Vol 68 (7) ◽  
pp. 515-529 ◽  
Author(s):  
Cecilia Lindskog ◽  
Max Backman ◽  
Agata Zieba ◽  
Anna Asplund ◽  
Mathias Uhlén ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Lines ◽  
Expression Patterns ◽  
Basic Research ◽  
Tissue Microarrays ◽  
Cancer Diagnostics ◽  
Proximity Ligation Assay ◽  
Mrna Levels ◽  
Proximity Ligation ◽  
Antibody Validation

Immunohistochemistry (IHC) is the accepted standard for spatial analysis of protein expression in tissues. IHC is widely used for cancer diagnostics and in basic research. The development of new antibodies to proteins with unknown expression patterns has created a demand for thorough validation. We have applied resources from the Human Protein Atlas project and the Antibody Portal at National Cancer Institute to generate protein expression data for 12 proteins across 39 cancer cell lines and 37 normal human tissue types. The outcome of IHC on consecutive sections from both cell and tissue microarrays using two independent antibodies for each protein was compared with in situ proximity ligation (isPLA), where binding by both antibodies is required to generate detection signals. Semi-quantitative scores from IHC and isPLA were compared with expression of the corresponding 12 transcripts across all cell lines and tissue types. Our results show a more consistent correlation between mRNA levels and isPLA as compared to IHC. The main benefits of isPLA include increased detection specificity and decreased unspecific staining compared to IHC. We conclude that implementing isPLA as a complement to IHC for analysis of protein expression and in antibody validation pipelines can lead to more accurate localization of proteins in tissue.

scholarly journals ADAM-17/FHL2 colocalisation suggests interaction and role of these proteins in colorectal cancer

Tumor Biology ◽  
2017 ◽  
Vol 39 (3) ◽  
pp. 101042831769502 ◽  
Author(s):  
Laurine Verset ◽  
Joke Tommelein ◽  
Christine Decaestecker ◽  
Elly De Vlieghere ◽  
Marc Bracke ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Cancer Progression ◽  
Colorectal Adenocarcinoma ◽  
Normal Mucosa ◽  
Scaffolding Protein ◽  
Proximity Ligation Assay ◽  
Low Grade ◽  
Proximity Ligation ◽  
Sw480 Cells

FHL2 is a multifunctional scaffolding protein; its expression is associated with poor prognosis in colorectal cancer. ADAM-17 is a metalloprotease implicated in ectodomain shedding. FHL2 regulates ADAM-17 plasma membrane localisation, and FHL2 deficiency leads to decreased activity of ADAM-17 in mouse macrophages. Presence and relationship of the ADAM-17/FHL2 complex with colorectal cancer progression is unknown. We studied FHL2 and ADAM-17 expression in several colon cancer cell lines by immunocytochemistry and western blot. To highlight the interaction between both molecules, we used the Duolink® kit for proximity ligation assay on SW480 cells. We also performed proximity ligation assay on biopsies and surgical specimens of colorectal adenocarcinoma and on matched normal mucosa. Furthermore, biopsies of colorectal adenoma with matched normal mucosa were selected. For quantification, pictures of the malignant, adenomatous and normal tissues were taken. Proximity ligation assay signals were quantified. Mean numbers of proximity ligation assay signals and of proximity ligation assay signals/nucleus were calculated. All cell lines showed FHL2 immunoreactivity; strongest positivity was observed in SW480 cells. ADAM-17 was expressed in all cell lines. Proximity ligation assay signals were present in SW480 cells. Quantitative analysis revealed that the interaction between FHL2 and ADAM-17 is more frequent in malignant than in normal tissue (p = 0.005). The mean number of ADAM-17/FHL2 proximity ligation assay signals was higher in colorectal adenocarcinoma than in adenoma with low-grade dysplasia (p = 0.0004). FHL2 interacts with ADAM-17 in normal, dysplastic and malignant colon epithelial cells. Colocalisation of these proteins is more frequent in malignant than in normal and dysplastic cells, suggesting a role for ADAM-17/FHL2 complex in the development of colorectal cancer.

scholarly journals Glyceraldehyde-3-Phosphate Dehydrogenase and Seven in Absentia Homolog -1 Interactions in Mammalian Cells Visualized Using Proximity Ligation Assay

2016 ◽  
Author(s):  
Harkewal Singh ◽  
Christopher Melm
Keyword(s):  
Cell Lines ◽  
Protein Interactions ◽  
Genetic Modification ◽  
Rolling Circle Amplification ◽  
Target Protein ◽  
Proximity Ligation Assay ◽  
Protein Protein Interactions ◽  
Proximity Ligation ◽  
Mammalian Cell Lines ◽  
Seven In Absentia

AbstractProteins seldom function in isolation and thus protein-protein interactions are critical in understanding the molecular basis of diseases and health (1, 2). There are several well established techniques that are used to investigate protein-protein interactions(3). Most of the methods require some form of genetic modification of the target protein and thus always adds extra steps. However, Proximity Ligation Assay(4-6) (PLA) aka Duolink® is one such method that requires no genetic modification of the target protein and probes protein-protein interactions in fixed live cells and tissues. Briefly, PLA requires the use of primary antibodies specific to the proteins of interest. Once the sample (fixed cells or tissues) is incubated with species specific primary antibodies, secondary antibodies that are conjugated with oligonucleotides (also known as PLUS and MINUS probes respectively) and connecter oligonucleotides are added. This complex is ligated if the two PLUS and MINUS probes are within 40nm of each other. The resulting nucleic acid is amplified using rolling circle amplification and then probed with appropriate fluorescent probes. If the two proteins are interacting, one could visualize the interaction as a single red foci (for example Far Red Detection) using fluorescent microscopy. Here, we used PLA to probe protein-protein interactions between Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and seven in absentia homolog -1 (Siah-1) – an E3 ubiquitin ligase. We first use PLA to show that GAPDH and Siah-1 proteins exist endogenously in the cytosol of multiple mammalian cell lines. Our data suggest the use of DU145 and T98G cell lines to show translocation of the GAPDH-Siah- 1 complex. Next, we used common nitrosylation agents(7, 8) (S-nitrosoglutathione-GSNO and S-Nitroso-N-acetyl-DL-pencillamine–SNAP) in different concentrations and observed that GAPDH and Siah-1 interact presumably due to the nitrosylation of the former, which is consistent with previous studies(9, 10). Interestingly, no interactions were observed between the two proteins in the absence of GSNO or SNAP indicating that nitrosylation might be critical for GAPDH-Siah1 interactions. Our results suggest that GAPDH-Siah-1 interactions originate in the cytosol and migrate to the nucleus under the conditions tested. We quantify the PLA signal using Duolink® Image Tool and observe a clear enhancement of GAPDH-Siah-1 PLA signal upon treating the cells with GSNO or SNAP. Next, we used R-(-)-Deprenyl (deprenyl), a known inhibitor of GAPDH4, and show that it abrogates GAPDH-Siah-1 PLA complex under the conditions tested. Finally, our data suggest that PLA can detect and quantify the GAPDH-Siah1 complex; a well-known protein-protein interaction implicated in neurodegeneration(9-11) and thus could be a method of choice for similar applications.

Significance of the IL-3 Receptor Beta Chain (IL-3Rbc) for FLT3-ITD Dependent Oncogeneic Signaling in AML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3660-3660
Author(s):  
Christoph Rummelt ◽  
Sivahari Prasad Gorantla ◽  
Philip Keye ◽  
Kathrin Klaesener ◽  
Venkatesh Rao ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Direct Interaction ◽  
Proximity Ligation Assay ◽  
Secondary Resistance ◽  
Knock Down ◽  
Proximity Ligation ◽  
Interaction Studies ◽  
Flt3 Mutations ◽  
Tki Resistance

Abstract Introduction Activating FLT3 mutations are found in 30% of AML patients. Internal tandem duplication (ITD) mutations are most common, and are associated with poor prognosis. FLT3 tyrosine kinase inhibitors (TKI) were shown to be effective in clinical trials. However, complete remissions are rare, responses are short-lived, and the majority of patients display primary or secondary resistance to FLT3 inhibition. In FLT3 kinase inhibitor resistant, FLT3-ITD positive cell lines, we identified a direct interaction between FLT3-ITD and IL-3Rβc. We therefore sought to characterize this interaction and determine its' role in FLT3-ITD oncogenic signaling. Methods Drug sensitive BA/F3 FLT3-ITD cells and sublines resistant to FLT3 TKIs were subjected to analysis of activated IL-3Rβc - JAK1/2 - STAT signaling by PCR, Western-Blot (WB) and Immunoprecipitation (IP). Interaction studies were performed in vitro using IVT FLT3-ITD and GST IL-3Rβc, and in cell lines using γ2A cells transfected with human FLT3-ITD and human IL-3Rβc, human MOLM13 and MV4-11 AML cell lines, and primary AML patient samples by WB, IP and proximity ligation assay (PLA). Mapping experiments were performed in mouse embryonic fibroblasts (MEF) transduced with FLT3-ITD and flag-IL-3Rβc full-length or cytoplasmic Y to F mutant constructs. Knockdown-experiments were performed in BA/F3 cells with stably transfected FLT3-ITD and inducable IL-3Rβc shRNA. Results In TKI resistant FLT3-ITD positive cell-lines that did not harbor secondary FLT3 mutations inducing TKI resistance, we observed phosphorylation of IL-3Rβc in 5/16 resistant lines (30%) in the presence of FLT3 TKI. In these cells, IL-3Rβc phosphorylation was mediated by an activating JAK1 V658F mutation that bypasses FLT3-ITD dependent IL-3Rβc phosphorylation. Thus, IL-3Rβc in FLT3-ITD expressing cells mediates JAK1/2-dependent TKI resistance. Of note in inhibitor sensitive cells, IL-3Rβc interacted with and was phosphorylated by FLT3-ITD in a JAK1/2 independent manner, suggesting that IL-3Rβc participates in FLT3-ITD dependent oncogeneic signaling. Indeed, in IL-3Rβc and JAK2 deficient γ2A cells, expression of human FLT3-ITD was sufficient to induce interaction with and phosphorylation of human IL-3Rβc. In human FLT3-ITD-positive AML cell lines MOLM13 and MV4-11, IL-3Rβc phosphorylation occurred in a FLT3-ITD dependent fashion and IL-3Rβc interacted with FLT3-ITD. Proximity ligation assay (PLA) experiments detected FTL3-ITD and IL-3Rβc in close proximity suggesting a direct interaction of both proteins in MOLM13 and MV4-11 cells, as well as in primary cells from FLT3-ITD positive AML patients. Interaction studies revealed that binding to FLT3-ITD occurred independent of IL-3Rβc cytoplasmic tyrosines, and phosphorylation of IL-3Rβc by FLT3-ITD did not require the presence of IL-3Rαc. Preliminary results from knock down experiments in FLT3-ITD expressing BA/F3 cells indicated that knock-down of IL-3Rβc renders cells more susceptible to FLT3 TKI inhibition. Conclusion These data suggest an entirely novel model of FLT3-ITD "physiologically" employing IL-3Rβc as signaling intermediate, and IL-3Rβc serving as a signaling module mediating JAK1/2-dependent TKI resistance. These findings point toward the significance of IL-3Rβc for FLT3-ITD dependent transformation and treatment resistance, supporting the relevance of IL-3Rβc as a possible treatment target in FLT3-ITD positive AML. Disclosures von Bubnoff: Novartis: Research Funding; BMS: Speakers Bureau.

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