Biology subjects

Brenner, W.

Publications and source records attributed to Brenner, W..

4 recordsLinked to original sources

bioRxiv2025

Rapid proteasomal degradation of mutant feline McDonough sarcoma-like tyrosine kinase-3 overcomes tyrosine kinase inhibitor resistance of acute myeloid leukemia cells

BackgroundFeline McDonough sarcoma (FMS)-related receptor tyrosine kinase 3 with activating internal tandem duplications (FLT3-ITD) causes acute myeloid leukemia (AML). Targeted protein degraders for FLT3 have evolved as drugs against leukemia. MethodsWe synthesized and characterized MA191 as novel von Hippel-Lindau (VHL)-based proteolysis targeting chimera (PROTAC) for FLT3. We analyzed protein expression, protein degradation mechanisms, and posttranslational modifications by immunoblot. Selective proteasome modulation, an inactive stereoisomer of MA191, and siRNA confirmed the event-driven degradation of FLT3-ITD. Hematopoietic cell survival and differentiation were determined by flow cytometry using apoptosis and cell surface markers. As models, we used cultured and primary human AML cells, FLT3 inhibitor-resistant AML cells, mature blood cells, and hematopoietic stem cells. We scrutinized the databases DepMap, GEPIA2, Hemap, and HPA to assess FLT3 expression and patient survival. Experiments with Danio rerio larvae verified in vivo anti-leukemic activity of MA191. ANOVA and Bonferroni correction were used for statistics. ResultsMA191 is a rapid nanomolar apoptosis inducer in AML cells harboring FLT3-ITD (IC50=10.16-11.6 nM; EC50=0.015-0.883 {micro}M). A stereoisomer of MA191 that cannot recruit VHL demonstrates that elimination of FLT3-ITD is superior to its inhibition. MA191 abrogates FLT3 inhibitor resistance from rebound activation of mitogen-activated kinases. Rapid depletion of FLT3-ITD by MA191 (DC50=10 nM) requires VHL, neddylation, and the pro-apoptotic BH3-only protein BIM. Reduction of FLT3-ITD by MA191 precedes apoptosis. This reveals an apoptosis-independent function of BIM on protein stability. Leukemia cells express more FLT3 than healthy cells (n=3675/n=1249) and FLT3 expression is associated with worse AML patient survival (p=0.0099). MA191 does not harm blood cells and bone marrow progenitor cells and does not disturb myeloid blood cell differentiation. In Danio rerio, MA191 halts AML cell proliferation without significant toxicity. Anti-leukemic effects of MA191 are not susceptible to anti-apoptotic effects of human stromal cells and mutations in the tyrosine kinase of FLT3-ITD that confer resistance to selective FLT3 inhibitors. Conclusionsthese insights and the disclosure of the structure of MA191 provide a framework for an improved design of PROTACs that target mutant FLT3 and are not vulnerable to extrinsic and intrinsic resistance mechanisms. Degradation kinetics appear as determinant of such resistance breakers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/660791v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@17e5988org.highwire.dtl.DTLVardef@27fbc4org.highwire.dtl.DTLVardef@1023648org.highwire.dtl.DTLVardef@39bdcb_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2019

CMKLR1-targeting peptide tracers for PET/MR imaging of breast cancer

Molecular targeting remains to be a promising approach in cancer medicine. Knowledge about molecular properties such as overexpression of G protein-coupled receptors (GPCRs) is thereby offering a powerful tool for tumor-selective imaging and treatment of cancer cells. We utilized chemerin-based peptides for CMKLR1 receptor targeting in a breast cancer xenograft model. By conjugation with radiolabeled chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), we obtained a family of highly specific and affine tracers for hybrid in vivo imaging with positron emission tomography (PET)/ magnetic resonance (MR) and concomitant biodistribution studies.\n\nMethodsWe developed five highly specific and affine peptide tracers targeting CMKLR1 by linker-based conjugation of chemerin peptide analogs (CG34 and CG36) with radiolabeled (68Ga) chelator DOTA. Our established xenograft model with target-positive DU4475 and negative A549 tumors in immunodeficient nude mice enabled CMKLR1-specific imaging in vivo. Therefore, we acquired small animal PET/MR images, assessed biodistribution by ex vivo measurements and investigated the tracer specificity by blocking experiments.\n\nResultsThe family of five CMKLR1-targeting peptide tracers demonstrated high biological activity and affinity in vitro with EC50 and IC50 values being below 2 nM. Our target-positive (DU4475) and target-negative (A549) xenograft model could be confirmed by ex vivo analysis of CMKLR1 expression and binding. After preliminary PET imaging, the three most promising tracers 68Ga-DOTA-AHX-CG34, 68Ga-DOTA-KCap-CG34 and 68Ga-DOTA-ADX-CG34 with apparent DU4475 tumor uptake were further analyzed. Hybrid PET/MR imaging along with concomitant biodistribution studies revealed distinct CMKLR1-specific uptake (5.1% IA/g, 4.5% IA/g and 6.2% IA/g 1 h post-injection) of our targeted tracers in DU4475 tumor tissue. More strikingly, the tumor uptake could be blocked by excess of unlabeled peptide (6.4-fold, 7.2-fold and 3.4-fold 1 h post-injection) and further confirmed the CMKLR1 specificity. As our five tracers, each with particular degree of hydrophobicity, showed different results regarding tumor uptake and organ distribution, we identified these three tracers with moderate, balanced properties to be the most potent in receptor-mediated tumor targeting.\n\nConclusionWith the breast cancer cell line DU4475, we established a model endogenously expressing our target CMKLR1 to evaluate our chemerin-based peptide tracers as highly affine and specific targeting agents. Eventually, we demonstrated the applicability of our 68Ga-labeled tracers by visualizing CMKLR1-positive breast cancer xenografts in PET/MR imaging and thus developed promising theranostics for tumor treatment.

cancer biology↗

bioRxiv2019

Increasing molar activity by HPLC purification improves 68Ga-DOTA-NAPamide tumor accumulation in a B16/F1 melanoma xenograft model

PurposeMelanocortin receptor 1 is overexpressed in melanoma and may be a molecular target for imaging and peptide receptor radionuclide therapy. 68Gallium labeling of DOTA-conjugated peptides is an established procedure in the clinic for use in positron emission tomography imaging. Aim of this study was to compare a standard labeling protocol against the 68Ga-DOTA peptide purified from the excess of unlabeled peptide. ProceduresThe MC1R ligand DOTA-NAPamide was labeled with 68Ga using a standard clinical protocol. Radioactive peptide was separated from the excess of unlabeled DOTA-NAPamide by HPLC. Immediately after the incubation of peptide and 68Ga (95 {degrees}C, 15 min), the reaction was loaded on a C18 column and separated by a water/acetonitrile gradient, allowing fractionation in less than 20 minutes. Radiolabeled products were compared in biodistribution studies and PET imaging using nude mice bearing MC1R-expressing B16/F1 xenograft tumors. ResultsIn biodistribution studies, the non-purified 68Ga-DOTA-NAPamide did not show significant uptake in the tumor at 1 h post injection (0.78% IA/g). By the additional HPLC step, the molar activity was raised around 10,000-fold by completely removing unlabeled peptide. Application of this rapid purification strategy led to a more than 8-fold increase in tumor uptake (7.0% IA/g). The addition of various amounts of unlabeled DOTA-NAPamide to the purified product led to a blocking effect and a decreased specific tumor uptake, similar to the result seen with non-purified radiopeptide. PET imaging was performed using the same tracers for biodistribution. Purified 68Ga-DOTA-NAPamide, in comparison, showed superior tumor uptake. ConclusionsWe demonstrated that chromatographic separation of radiolabeled from excess unlabeled peptide is technically feasible and beneficial, even for short-lived isotopes such as 68Ga. Unlabeled peptide molecules compete with receptor binding sites in the target tissue. Purification of the radiopeptide therefore improved tumor uptake.

cancer biology↗

bioRxiv2018

Non-canonical HIF-1 stabilization is essential for intestinal tumorigenesis

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1s role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of wnt/{beta}-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with nuclear HIF-1 in intestinal adenomas, pointing towards a functional importance of hypoxia-independent, i.e. non-canonical HIF-1 stabilization that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1 stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

cancer biology↗