However, the lack of specific agonists for all α1 subtypes has limited our understanding of the physiological roles of different α1-AR subtypes, and led to the stagnancy in agonist-based drug development for these receptors. Here we report cryo-EM structures of α1A-AR in complex with heterotrimeric G-proteins and either the endogenous common agonist epinephrine or the α1A-AR-specific synthetic agonist A61603. These structures provide molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by A61603. Guided by the structures and corresponding molecular dynamics simulations, we engineer α1A-AR mutants that are not responsive to A61603, and α1B-AR mutants that can be potently activated by A61603. Together, these findings advance our understanding of the agonist specificity for α1-ARs at the molecular level, opening the possibility of rational design of subtype-specific agonists.α α1-adrenergic receptors (α1- AR) play critical roles in the cardiovascular and nervous systems. Here, the authors report molecular insights into the mechanisms underlying the discrimination between α1A-AR and α1B-AR by the agonist A61603. They function through nine distinct human adrenergic receptor (AR) subtypes: α1-type , α2-type  and β-type 4. Due to the lack of selective pharmacological agonists for α1-ARs, the therapeutic potential of α1-ARs has been largely unexplored9. However, mouse genetic studies with individual gene deletions have demonstrated distinct but overlapping physiological functions of α1-AR subtypes in the regulation of blood pressure, cardiac hypertrophy, vascular smooth muscle contraction, neurotransmission, learning and memory, and metabolism17. For example, the α1A-AR subtype is a vasopressor expressed in resistance arteries and is required for normal arterial blood pressure regulation11. In addition, α1B-AR in human coronary endothelial cells mediates vasodilation19.Epinephrine and norepinephrine are neurotransmitters of the sympathetic nervous system, and hormones secreted by the adrenal medulla1-ARs couple to the Gq family of G-proteins, leading to the stimulation of phospholipase C-β to cleave phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate and 2-diacylglycerol20. The former promotes the release of Ca2+ from intracellular stores, while the latter activates protein kinase C22. While some pharmacological compounds exist that can target α1-ARs, there are only two structurally related α1A-specific agonists, and no α1B-AR and α1D-AR selective agonists have been reported17. A61603 is a high affinity, selective α1A-AR agonist which shows almost no activity at α1B-AR and α1D-AR23. Furthermore, there are no structures available for the active states of α1-ARs. In this paper, we use cryo-EM to determine the structures of α1A-AR/Gq signaling complexes with epinephrine or A61603. These structures reveal the molecular basis for the binding specificity of A61603 for α1A-AR, and the different conformations of epinephrine in interacting with α-ARs versus β-ARs. Gaussian accelerated molecular dynamics (GaMD) simulations and functional studies provide further insights into the mechanisms of specificity, ultimately enabling the validation of key sites that determine the ability of A61603 to specifically activate α1A-AR but not α1B-AR25.α1-AR, we solved cryo-EM structures of human α1A-AR bound to A61603 (a synthetic specific agonist for α1A-AR) at 2.6 Å or epinephrine  at 3.0 Å, both in complex with its cognate signaling Gq heterotrimer (mini-GαqGβ1Gγ2) 26. We thus compared our active state structures of α1A-AR with this inactive state structure of α1B-AR. This comparison revealed characteristic conformational changes associated with class A GPCR activation     denotes the opening of TM6 in class A GPCRs for G-protein engagement27 , and W6.48 to form new contacts  Fig. , and an 53) Fig. . In addi1A-AR–Gq, the para-hydroxyl group of the catechol ring of epinephrine forms H-bond with the hydroxyl side-chain of S1885.43  and N3127.38 (in β2-AR) interacted through hydrogen bonds with the β-carbon hydroxyl and the N-methyl group of epinephrine. In α1A-AR, it is F3127.38 at the equivalent position, and its side-chain did not form hydrogen bonds with epinephrine  in the methanesulfonamide group of A61603 in the GaMD simulations , α1A-AR(A189S), and α1A-AR(M292L) by mutating these residues to the corresponding residues in α1B-AR. Since these residues are involved in hydrophobic interactions with A61603 . Since α1A-AR is coupled to Gq, we used calcium responses as a functional readout  reduced the apparent affinity of the A61603 response by more than 1000-fold , α1A-AR(A189S), α1A-AR(M292L), and α1A-AR , α1A-AR(M292L), and α1A-AR relative to the starting cryo-EM structure were 4.62 ± 1.68 Å, 3.21 ± 1.05 Å, and 3.99 ± 1.13 Å, respectively , α1A-AR(M292L), and α1A-AR reduced the binding of A61603 to the receptor  exhibited a smaller RMSD with lower fluctuations in the GaMD simulations , α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were 4.25 ± 0.98 Å, 3.43 ± 0.47 Å, 4.13 ± 1.33 Å, and 4.38 ± 1.27 Å, respectively , α1A-AR(M292L), and α1A-AR, but still maintained in α1A-AR(A189S), during the simulations  and −11.52 ± 2.47 kcal/mol for α1A-AR, compared to −26.73 ± 0.22 kcal/mol for the wild-type α1A-AR. α1A-AR(A189S) exhibited a similar binding free energy of A61603  as the wild-type α1A-AR. However, an exception was observed in α1A-AR(M292L), where the binding free energy was −27.88 ± 1.53 kcal/mol. This likely resulted from the exclusion of the entropy and MM/GBSA free energy calculations of GaMD simulation frames without the energetic reweighting36. In general, the systems with a stronger functional response to A61603-binding exhibited more favorable binding free energy compared to those with weaker or no functional responses. Therefore, the GaMD simulation findings correlated well with the calcium response data.In parallel, GaMD simulations were performed on αα1A-ARA18S, α1A-AR, α1A-ARM92L, and 1B-AR from non-responsive to responsive to A61603. This type of exercise is usually difficult given that many residues are typically involved in ligand interactions. However, based on our above data, most of the agonist interacting residues are the same between α1A-AR and α1B-AR, except for the three residues . Wild-type α1B-AR did not respond to A61603 at concentrations up to 10 μM, while it was responsive to epinephrine  responded to A61603 in a dose-dependent manner with an EC50 around 10 nM , α1B-AR(S208A) or α1B-AR(L314M) alone did not respond to A61603 , and α1B-AR. In wild-type α1B-AR and α1B-AR(S208A), A61603 exhibited large RMSDs relative to the starting conformation, with reduced interactions between A61603 and α1B-AR  exhibited low RMSD at ~2.5 Å, forming stable interactions with residues D1253.32 and S2075.43 in the receptor  showed the strongest binding affinity , followed by α1B-AR(S208A)  and wild-type α1B-AR  , α1A-AR(V185A), α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were 1.46 ± 0.10 Å, 2.36 ± 0.52 Å, 1.10 ± 0.088 Å, 1.74 ± 0.39 Å, and 2.58 ± 0.33 Å, respectively (Supplementary Table 1A-AR(A189S)  exhibited a similar RMSF value as wild-type α1A-AR, while the other α1A-AR mutants  showed higher A61603 fluctuations. The RMSF values of A61603 in α1B-AR, α1B-AR(S208A) and wild-type α1B-AR were 0.98 ± 0.21 Å, 3.55 ± 1.97 Å, and 4.28 ± 0.30 Å, respectively , one low-energy state of A61603 with a narrow conformational space was sampled. Conversely, the other mutant α1A-ARs sampled larger conformational space with at least two distinct low-energy states. Among the α1B-AR systems, α1B-AR sampled only one low-energy state, while α1B-AR(WT) and α1B-AR(S208A) sampled at least two low-energy states with much larger conformational space  of A61603 in different receptor systems. In this context, larger RMSF values indicate weaker binding of A61603. The calculated RMSF values of A61603 in α, α1A-ARM92L, and 1-ARs are one of the three major subfamilies of ARs. Due to the lack of specific agonists, the physiological functions of α1-ARs are still not completely revealed, and their therapeutic potentials in treating cardiovascular, neurological, neuropsychiatric, and inflammatory disorders are not fully exploited. Here we have investigated the structural basis of activation and agonist interactions with α1-AR. Our data shows that M2926.55 and V1855.40 of α1A-AR define the specificity of A61603 for α1A-AR. Hydrophobic M2926.55 in α1A-AR corresponds to hydrophobic L6.55 in α1B-AR and α1D-AR, while aromatic Y6.55 in α2-AR and polar N6.55 in β-ARs  revealed that they share some conserved interactions while also displaying certain unique interactions  residues 228-255, and C-terminal residues 351-466. A hemagglutinin (HA) signal peptide, a FLAG tag, and a T4 lysozyme were fused to its N-terminus, and a PreScission protease cleavage site, an eGFP, and an 8xHis tag were added to its C-terminus. This construct was subcloned into pFastBac1 vector for expression in Spodoptera frugiperda Sf9 insect cells. Sf9 cells were grown to 2 to 3 million cells per ml in ESF 921 protein-free medium (Expression Systems) before 50 ml of baculoviruses were added for infection. 72 h later, cells were harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80 °C until use. For α1A-AR purification, thawed cell pellets were lysed with 1% n-Dodecyl-β-d-Maltopyranoside  in a buffer containing 25 mM HEPES (pH 7.0), 350 mM NaCl, 100 μM A61603  or epinephrine , supplemented with protease inhibitors  at 4 °C. Insoluble fractions were removed by ultracentrifugation at 142,000 × g for 1 h at 4 °C. The supernatant was then incubated with house-made GFP nanobody beads for at least 2 h, and washed with a buffer containing 25 mM HEPES (pH 7.0), 350 mM NaCl, 0.05% DDM, and 100 μM A61603 or epinephrine before PreScission protease was added to elute α1A-AR from GFP nanobody beads overnight at 4 °C. Eluted α1A-AR was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 25 mM HEPES (pH 7.0), 150 mM NaCl, 0.02% Lauryl Maltose Neopentyl Glycol , and 100 μM A61603 or epinephrine. Peak fractions were pooled and concentrated for complex assembly.The human αiN–Gβ1–Gγ2 and scFv16 were kindly provided by Dr. Bryan L Roth (University of North Caroline at Chapel Hill)37. For the expression of heterotrimeric mini-GαqiN–Gβ1–Gγ2, a single virus that encodes all three subunits, including mini-GαqiN, Gβ1, and Gγ2, with a 6xHis tag fused to the N-terminus of Gβ1, was used to infect Sf9 cells. 48 h post infection, cells were harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80 °C. Cell pellets were thawed in a lysis buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 2 mM β-mercaptoethanol, 1 mM MgCl2, 0.1 mM GDP, 0.2% (v/v) Triton X-100, supplemented with protease inhibitors  at 4 °C and insoluble fractions were removed by centrifugation at 142,000 × g for 1 h. Supernatant was collected and incubated with HisPur Ni-NTA Resin (Thermo Fisher Scientific) for 2 h at 4 °C. Resin was then washed with 20 mM imidazole in the lysis buffer, and eluted with 250 mM imidazole in the lysis buffer. Eluted heterotrimeric G-protein was further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.02% LMNG, 0.1 mM TCEP, 1 mM MgCl2, and 10 μM GDP. Peak fractions were pooled and concentrated for complex assembly.The expression constructs of heterotrimeric mini-Gαq37. 72 h after infection, Sf9 cell culture media expressing scFv16 was collected and PH balanced to pH 8.0 by addition of Tris buffer. 10 mM calcium chloride was then added to quench the chelating agents with stirring at room temperature for 1 h. The precipitate was removed by centrifugation at 8000 × g for 30 min. The supernatant was incubated with HisPur Ni-NTA Resin with stirring for 4 h at 4 °C. The Ni-NTA resin was then collected with centrifugation at 800 g and washed with 20 mM imidazole in a buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl before 200 mM imidazole in the same buffer was used to elute the protein. The C-terminal 8xHis tag was removed by the treatment of PreScission protease overnight at 4 °C. The protein was then further purified by size-exclusion chromatography using a Superdex 200 Increase 10/300 column  equilibrated with a buffer containing 20 mM HEPES (pH 7.5) and 100 mM NaCl. Peak fractions were pooled and concentrated for complex assembly.scFv16 was expressed and purified from Sf9 cells as a secreted protein1A-AR–Gq–scFv16 complex, α1A-AR, and heterotrimeric Gq were mixed at a 1:1.5 molar ratio and incubated for 30 min at room temperature followed by the treatment of 0.4 U Apyrase (New England Biolabs) for another 30 min at room temperature. A 1.5 molar access of scFv16 was then added to the mixture and incubated overnight at 4 °C. The next day, the mixture was centrifuged at 16,000 × g for 10 min to remove any precipitants. The supernatant was then loaded onto a Superdex 200 Increase 10/300 column equilibrated with 10 mM HEPES pH 7.0, 100 mM NaCl, 0.1 mM TCEP, 0.02% LMNG, and 30 μM A61603 or epinephrine. The elution fractions from a single peak containing pure α1A-AR–Gq–scFv16 complex concentrated to 2.5 mg/ml and used directly for making cryo-EM grids.To assemble the α1A-AR–Gq–scFv16 complex at a concentration of 2.5 mg/ml was applied to glow-discharged 400 mesh gold Quantifoil R1.2/1.3 holey carbon grids (Quantifoil Micro Tools) and vitrified using a Vitrobot Mark IV (Thermo Fisher Scientific/FEI) at 22 °C and 100% humidity. Micrographs were collected on a 300 kV Titan Krios electron microscope (Thermo Fisher Scientific/FEI) with a Gatan K3 direct electron detector  in super-resolution mode at a nominal ×81,000 and ×64,000 magnification for the A61603 and epinephrine-bound complexes, respectively. For the A61603-bound complex, 7,773 movies in the defocus range of −1.0 to −1.8 μm were recorded with a total accumulated dose of 50 e−/Å2. For the epinephrine-bound complex, 10,486 movies in the defocus range of −1.0 to −1.8 μm were recorded with a total accumulated dose of 52 e−/Å2.3.5 µL of α44. The effects of contrast-transfer function were estimated with CTFFIND v4.1.845. For the A61603 dataset, Relion 2D template-based auto-picking was used to pick particles and the resulting particle stacks were two-times Fourier cropped and processed through 2D classification in CryoSparc v3.3.1 to remove false positives, receptor alone, or G-proteins alone particles46  and 219,834 (epinephrine) particles obtained by 3D variability analysis that yielded higher resolution in the transmembrane region were selected and non-uniform refinement of these particles in CryoSparc yielded maps with 2.6 Å-resolution (A61603) and 3.0 Å-resolution (epinephrine) using the 0.143 Fourier Shell Correlation criterion. These consensus stacks were also subjected to focused refinement of α1A-AR and Gαq region in CryoSparc. The focused refinement map showed significant improvement in the α1A-AR region compared to the consensus map. Models were built starting from AlphaFold predicted structure of inactive α1A-AR (AF-P35348-F1) and scFv16 bound Gq heterotrimer (PDB code 6WHA) in Coot v0.9.1, which combined with consensus and focused refinement maps, were used to generate composite maps in Phenix dev-469448. The resulting maps were super-sampled in Coot to 0.856 Å per pixel (A61603) and 0.8608 Å (epinephrine) with 320-voxel boxes. The A61603 and epinephrine bound α1A-AR–Gq–scFv16 models were real-space refined against the composite maps in Phenix, and a work/free half-map pair was used to ensure against over-fitting  were added by SWISS-MODEL51. The initial models of the A61603-bound mutants of α1A-AR(V185A), α1A-AR(A189S), α1A-AR(M292L), and α1A-AR were built by VMD based on the A61603–α1A-AR–Gq cryo-EM structure. All six simulation systems were prepared with the CHARMM-GUI web server for using the membrane protein input generator52. The receptor was inserted into a palmitoyl-oleoyl-phosphatidyl-choline (POPC) bilayer. All chain termini were capped with neutral patches (acetyl and methylamide). All the disulfide bonds in the complexes that were resolved in the cryo-EM structures were maintained in the simulations. The systems were solvated in 0.15 M NaCl solution at temperature 310 K. The AMBER ff14SB and AMBER LIPID 21 parameter sets were used for the receptor and lipids54. The GAFF2 parameters with RESP charges were used for epinephrine and A6160355. For each of the complex systems, initial energy minimization, thermalization, and 20 ns cMD equilibration were performed before GaMD simulations. A cutoff distance of 9 Å was used for the van der Waals and short-range electrostatic interactions and the long-range electrostatic interactions were computed with the particle-mesh Ewald summation method56. A 2-fs integration time step was used for all MD simulations. The SHAKE algorithm was applied to all hydrogen-containing bonds. With all other atoms fixed, the lipid tails were energy minimized for 5000 steps using the conjugate gradient algorithm and melted with a constant number, volume, and temperature (NVT) run for 0.5 ns at 310 K. The six systems were further equilibrated using a constant number, pressure, and temperature (NPT) run at 1 atm and 310 K for 1 ns with 1.0 kcal/(mol Å2) harmonic position restraints applied to the protein and ligand atoms. Then, conventional MD simulations were performed on each system for 20 ns at 1 atm pressure and 310 K with a constant ratio constraint applied on the lipid bilayer in the X-Y plane. The GaMD module implemented in the GPU version of AMBER22 was then applied to perform the simulations57. GaMD simulations included an 8-ns short cMD simulation used to collect the potential statistics for calculating GaMD acceleration parameters, and a 48-ns equilibration after adding the boost potential. Finally, three independent 500-ns GaMD simulations with randomized initial atomic velocities were performed for the epinephrine–α1A-AR–Gq, A61603–α1A-AR–Gq, and A61603-bound mutant α1A-AR–Gq complexes. The average and SD of the system potential energies were calculated every 800,000 steps (1.6 ns). All GaMD simulations were run at the “dual-boost” level by setting the reference energy to the lower bound. One boost potential was applied to the dihedral energetic term and the other to the total potential energetic term. The upper limit of the boost potential SD, σ0 was set to 6.0 kcal/mol for both the dihedral and the total potential energetic terms.The epinephrine–α58. Root-mean-square fluctuations (RMSFs) were calculated for the agonist A61603, averaged over three independent GaMD simulations. Interactions between epinephrine and the receptor were measured by the distance between the CG atom of D106 and N1 atom of epinephrine, and the OG atom of S188 and O2 atom of epinephrine. Interactions between A61603 and the receptor were measured by the distance between the CG atom of D106 and the N2 atom of A61603, the N atom of A189 and the O2 atom of A61603, the OG atom S188 and the N atom of A61603, the OG atom of S188 and the O atom of A61603, and the OG atom of S188 and the O2 atom of A61603. Time courses of these reaction coordinates obtained from the GaMD simulation were plotted in Fig. CPPTRAJ was used to analyze the GaMD simulations2 at 37oC in humidified incubator. HEK293T cells were seeded on poly-L-lysine coated glass coverslips (18 mm) one day before transfection. GCaMP 6 f (0.2 μg per well) and α1A-AR plasmid (0.5 µg per well) were used to transfect cells using Lipofectamine 2000 in 12-well plates. Cells were protected with 10 μM Prazosin 6–8 hours after transfection. Calcium imaging experiments were performed 24 hours after transfection. Cells were placed on an inverted Nikon Eclipse Ti2-E microscope equipped with an Andor Zyla 5.5 sCMOS camera, Lumencor SOLA-SE II light engine, and CFI Nikon Plan Apo Lambda ×20 with 0.75 NA. GCaMP6f was excited using 488 nm SOLA LED light. Cells were continuously perfused with extracellular (EX) solution (containing (in mM): 135 NaCl, 5.4 KCl, 10 HEPES, 2 CaCl2, 1 MgCl2, pH = 7.4) utilizing a gravity-driven perfusion system at room temperature. Drugs (A61603 or epinephrine) were dissolved in EX solution. Calcium imaging was analyzed using ImageJ 1.52a and NIS-Elements Advance Research 5.2.6. Each single cell was manually selected as region of interest and the fluorescence intensity was normalized to the baseline before agonist application. An averaged trace (60–200 cells per recording) was used to quantify maximum fluorescence amplitude after drug application. Dose–response curve was done using nonlinear fit in GraphPad Prism.HEK293T cells were purchased from ATCC , authenticated by Bio-Synthesis, Inc. and routinely tested negative for mycoplasma. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) in 5% CO2+ assays were repeated three times, and the data are represented as mean ± SD of the three independent experiments. The analysis was done using the log(agonist) vs. response function of Prism 9 (GraphPad) as indicated in the figure legends. Cryo-EM data collection and refinement statistics are listed in Supplementary Table In Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


Multiple prognostic biomarkers, including circulating tumour cell (CTC) counts, exist in metastatic castration‐resistant prostate cancer (mCRPC) patients, but none of them have been implemented into daily clinical care. The modified fast aneuploidy screening test‐sequencing system (mFast‐SeqS), which yields a genome‐wide aneuploidy score, is able to reflect the fraction of cell‐free tumour DNA (ctDNA) within cell‐free DNA (cfDNA) and may be a promising biomarker in mCRPC. In this study, we investigated the prognostic value of dichotomized aneuploidy scores (< 5 vs. ≥ 5) as well as CTC counts (< 5 vs. ≥ 5) in 131 mCRPC patients prior to treatment with cabazitaxel. We validated our findings in an independent cohort of 50 similarly treated mCRPC patients. We observed that, similar to the dichotomized CTC count , dichotomized aneuploidy scores  significantly correlated with overall survival in mCRPC patients. We conclude that a dichotomized aneuploidy score from cfDNA is a prognostic marker for survival in mCRPC patients within our discovery cohort and in an independent mCRPC validation cohort. Therefore, this easy and robust minimally‐invasive assay can be readily implemented as a prognostic marker in mCRPC. A dichotomized aneuploidy score might also be used as a stratification factor in clinical studies to account for tumour load. The dichotomized mFast‐SeqS‐based aneuploidy score from cfDNA is a prognostic marker for survival in mCRPC patients. This easy, robust, low‐cost, and minimally invasive assay can be readily implemented as a prognostic marker in mCRPC. This assay represents an attractive stand‐alone stratification tool for use in daily clinical practice, and could also aid as a stratification factor in clinical studies. ALBalbumin levelsANCabsolute neutrophil countcfDNAcell‐free DNACIconfidence intervalctDNAcell‐free tumour DNAEpCamepithelial cell adhesion moleculeHBGhemoglobin levelsHRhazard ratioLDHlactate dehydrogenase levelsmCRPCmetastatic castration‐resistant prostate cancermFast‐SeqSmodified Fast Aneuploidy Screening Test‐Sequencing SystemNIPTnoninvasive prenatal testOSoverall survival1The daily clinical care of metastatic castration‐resistant prostate cancer (mCRPC) patients could be improved by utilizing non‐invasively derived prognostic markers. Circulating tumour cells (CTCs) and circulating tumour‐derived DNA (ctDNA) comprise two minimally invasive and safely obtainable biomarkers from liquid biopsies . ExtensiIn this manuscript, we investigated the prognostic value of mFast‐SeqS‐derived dichotomized aneuploidy scores for mCRPC patients and compared their performance to dichotomized CTC counts.22.1et al. [For our discovery cohort, we included 131 out of 137 mCRPC patients with known CTC counts from the CABA‐V7 trial (MEC16‐703), as previously detailed by Isebia et al. . Six patn = 50) with known CTC counts from the CABARESC trial , as previously detailed by Nieuweboer et al. [U test and Fisher's exact test) where we used P > 0.1 to propose no difference.As an independent validation cohort, we selected mCRPC patients  contained sufficient events.The outcome of interest was overall survival (OS), as measured from the inclusion of the study until death from any cause. Based on results in our discovery cohort, we calculated that in order to have 80% power to detect a similar HR in the validation cohort given a two‐sided α of 5%, a minimum of 32 events (i.e. deaths) needed to be observed. The included validation cohort  according to the manufacturer's instructions. Briefly, CTC enumeration was performed on 7.5 mL of CS blood using the Circulating Epithelial Cell Kit on the CellSearch system. Using ferrofluid labelled anti‐EpCAM antibodies, immunomagnetically captured cells are characterized using different combinations of staining reagents.As previously detailed by Isebia et al.  and Onst [et al.  for the 2.3et al. [g, respectively. EDTA tubes were processed within 24 h and CS tubes within 96 h, respectively. Plasma was then immediately stored at −80 C in 2 mL aliquots until cfDNA isolation. Next, cfDNA was isolated using the QIAamp® Circulating Nucleic Acid kit  and Maxwell® RSC ccfDNA Plasma Kit  according to the manufacturer's instructions.Per included patient (CABA‐V7 and CABARESC), collected blood was centrifuged twice to obtain plasma, as detailed by van Dessel et al. , and pla2.4et al. [Z‐score that serves as an estimate for overall aneuploidy. As described by Belic et al. [m) and supplemented with a 5% PhiX control library. The libraries were sequenced single‐ended for 150 bp reads on a MiSeq‐sequencer . Samples yielding < 90 000 reads were sequenced again, and resulting reads were combined [We employed the mFast‐SeqS platform on 1 ng of cfDNA to obtain low‐resolution copy‐number profiles. Aneuploidy scores per chromosome arm were determined using the mFast‐SeqS method. The original Fast‐SeqS  was deveet al.  to estimc et al. , we usedcombined .trimmomatic [Z‐score  from Belic et al.) [Z‐scores per chromosome arm were squared and summed into a genome‐wide aneuploidy score per patient. Samples with a genome‐wide Z‐score ≥ 5   trimmoma2.5et al. [Using the maximum variant allele frequencies per sample as derived from our targeted QIASeq panel as detailed by Isebia et al. , we perf2.6cutpointr package (version 1.1.2) [n = 131) based on their survival status ; using 10.000 bootstrap iterations.We utilized the n 1.1.2)  to estab2.7n = 131) for predicting OS, as measured from inclusion of study until death from any cause, were studied in a univariate Cox proportional hazards regression analysis. The following characteristics were investigated: age at registration, total Gleason‐score, PSA at primary diagnosis, dichotomized WHO‐status (0 vs. 1–2), albumin levels (ALB), absolute neutrophil count (ANC), haemoglobin levels (HBG), number of white blood cells (WBC), alkaline phosphatase levels (ALP), lactate dehydrogenase levels (LDH), dichotomized aneuploidy scores at baseline (aneuploidy score < 5 vs. ≥ 5) and dichotomized number of CTCs (per 7.5 mL) at baseline (< 5 vs. ≥ 5 CTCs). We only retained characteristics with P < 0.1 for subsequent multivariable Cox proportional hazards regression where backward selection was applied with a threshold of P < 0.1. In addition, sample‐specific dichotomized aneuploidy scores and dichotomized CTC counts were combined to identify potential complimentary effects. Kaplan–Meier curves were generated for dichotomized aneuploidy score, dichotomized CTC count and dichotomized WHO status and obtained curves were tested using the log‐rank test.The most relevant baseline characteristics from the discovery cohort (r (version 4.2.1).All analyses were performed on the statistical language platform 33.1n = 131; 92 deaths; Fig. n = 50) and the complete CABARESC study group (n = 224) from which the validation cohort was drawn  and dichotomized CTC counts (< 5 or ≥ 5) were analysed for predicting OS using Cox proportional hazards regression analysis including all relevant clinical characteristics (based on backward selection). This revealed that dichotomized aneuploidy scores and dichotomized CTC counts both independently served as significant non‐invasive biomarkers predicting OS (Our previous threshold for the dichotomization of mFast‐SeqS scores (< 5 or ≥ 5) was based on previous studies , 11, 21.4Numerous efforts and studies have been undertaken to study and improve the prognostic value of non‐invasively derived markers in mCRPC patients , 24, 25.et al. [Different methods exist for detecting ctDNA within the total pool of cfDNA. These ctDNA analyses are variable in their design; some focusing on single tumour‐specific gene alterations, whilst others, for example, quantify ctDNA by utilizing aneuploidy detection based on copy number alterations  or singlet al.  showed tThe exploitation of dichotomized aneuploidy scores as prognostic markers for mCRPC patients has not yet been investigated, whilst it could complement CTC enumeration. In this analysis, we investigated the prognostic value of aneuploidy scores for mCRPC patients and compared their performance to dichotomized CTC counts.We observed a prognostic value for OS for cfDNA‐based dichotomized aneuploidy scores. These findings were significant in our discovery cohort (CABA‐V7) and were replicated in our comparable mCRPC validation cohort (CABARESC). Dichotomized aneuploidy scores and their combination with dichotomized CTC counts hold complementary benefits in identifying the mCRPC patients with the worst outcome. Additionally, we revealed that dichotomized aneuploidy scores and dichotomized CTC counts both independently serve as significant non‐invasive biomarkers for predicting OS.Despite these significant observations, this work has limitations. Our employed thresholds for dichotomization of CTC counts and aneuploidy scores are currently based on the previously employed threshold , 8, whic5In conclusion, the mFast‐SeqS‐derived aneuploidy score is a global, minimally invasive and user‐friendly assay that can be employed unrestrictedly without the use of specialized equipment such as CellSearch. In this study, we showed that a dichotomized aneuploidy score is a clinically relevant stratification marker, akin to an established CTC count. The mFast‐SeqS method can be used for an estimation of ctDNA percentage, since it correlates with tumour content and can be easily used for monitoring disease progression without prior knowledge of the genetic composition of the malignancy. The mFAST‐SeqS assay provides an intuitive low‐resolution copy number profile, requiring only a low input of 1 ng cfDNA and results can typically be obtained within 2 days. Therefore, this affordable assay represents an attractive stand‐alone stratification tool for usage in daily clinical practice, which could also aid as a stratification factor in clinical studies. Whether mFast‐SeqS‐derived aneuploidy score could also serve as a predictive biomarker for therapy response or longitudinal therapy monitoring should be further investigated.Ronald de Wit has acted in a consulting or advisory role for Sanofi, Merck, and Bayer and has received research funding from Bayer, Sanofi.Martijn P. Lolkema has acted in a consulting or advisory role for Sanofi, Johnson & Johnson, Merck, Astellas, Incyte, Amgen, Janssen Cilag, Bayer, Servier and Pfizer; and has received research funding from Sanofi, Astellas, Janssen Cilag and MSD.John. W. M. Martens has acted in a consulting or advisory role for Novartis and Roche; and has received research funding from Philips, Therawis, Pamgene, Cergentis, Cytotrack.Bianca Mostert has acted in a consulting or advisory role for Servier, Lilly and BMS; and has received research funding from Sanofi, Pfizer and BMS.Ron H.J. Mathijssen received research funding from Astellas, Bayer, Cristal Therapeutics, Novartis, Pamgene, Pfizer, Roche, Sanofi, Boehringer‐Ingelheim and Servier.All authors certify that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript , are the following:The remaining authors have nothing to disclose.KTI, JvR, JWMM and MPL had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: MPL, JWMM, BM, SMW, RdW. Acquisition of data: KTI, BM, TD, JK, VdW, EOH, PH, BCMH, HHH, RdW, RHJM, MPL, SMW, JvR, JWMM. Analysis and interpretation of data: KTI, JvR, MPL, SMW, TD, BM, EOH, RHJM, JWMM. Drafting of the manuscript: KTI, JvR, MPL. Critical revision of the manuscript for important intellectual content: KTI, BM, TD, JK, VdW, EOH, PH, BCMH, HHH, RdW, RHJM, MPL, SMW, JvR, JWMM. Statistical analysis: KTI, JvR, EOH. Obtaining funding: MPL, RdW, BM. Administrative, technical, or material support: KTI, JvR, SMW, TD, VdW, JK, BCMH, PH, HHH. Supervision: JWMM.https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/1878‐0261.13449.The peer review history for this article is available at Fig. S1. Association of aneuploidy scores with other molecular characteristics. (a) Aneuploidy scores  per dichotomized aneuploidy group. Boxplots represent the median and first and third quantile whilst error bars depict the interquartile range (IQR)x1.5. (b) Aneuploidy scores  versus sample‐specific maximum variant allele frequency derived from a targeted QIASeq panel of 57 genes . Spearman correlation coefficient (ρ) and statistical significance of observed association (p), together with a linear model equation as depicted by a blue line with 95% confidence level interval as gray background, is shown in top. (c) Same a b) but for CTC counts .Click here for additional data file.Fig. S2. Overall survival versus alternative dichotomized aneuploidy scores. Survival probability (OS), as measured from inclusion of study until death from any cause, using univariate analysis of all patients per cohort (y‐axis), stratified and colored by varying dichotomized categories at baseline, depicted in months (x‐axis); censoring is shown by crosses (+). The bottom table represents the total number of remaining cases per depicted time‐point. The log‐rank p‐value, hazard ratio (death) with 95% CI and Akaike information criterion (AIC) is shown on the right‐hand top‐side. The 50% survival probabilities per strata as indicated by dashed lines whilst the confidence interval per stratum is indicated by transparent backgrounds. a) Discovery cohort (CABA‐V7), b) Validation cohort (CABARESC).Click here for additional data file.Fig. S3. Overall survival for all included patients in the discovery cohort, full CABARESC cohort and the included CABARESC cohort as validation cohort. Survival probability (OS) using univariate analysis of all included patients per cohort (y‐axis), stratified and colored by cohort, depicted in months (x‐axis); censoring is shown by crosses (+). The bottom table represents the total number of remaining cases per depicted time‐point. Hazard ratios with 95% confidence interval (CI) from multivariate Cox proportional hazards regression within the discovery cohort (n = 131). The p‐values for each multivariate assessment are presented on the right‐hand side of each comparison.Click here for additional data file.Table S1. Baseline characteristics of the complete CABARESC cohort and the included subset used for validation.Click here for additional data file.Table S2. Overview of included patients and data presented in figures. Overview of all data as presented and quantified in this manuscript.Click here for additional data file.


Triple negative breast cancer (TNBC) comprises 10–20% of diagnosed breast cancers. TNBCs are devoid of common biomarkers such as an estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Research is currently being conducted to determine the androgen receptor’s (AR) role in TNBC and determine its ability to be utilized as an effective drug target in the absence of the commonly targeted receptors. Many studies combine anti-androgen drugs with other chemotherapy to decrease tumor growth and proliferation. The further understanding of AR’s mechanism in tumor cells can improve drug efficacy as well as the prognoses of patients suffering from TNBC.To assess AR’s role in TNBC treatment, various existing and completed clinical trials targeting AR or co-targeting AR with other pertinent signaling molecules were analyzed. Cyclin-dependent kinase 4/6 (CDK4/6), cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17 lyase), and the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were some of the most prevalent biomarkers used in combination therapy with AR inhibitors in these trials. Studying how AR functions in tandem with these molecules can have increasing breakthroughs in the treatment options for TNBC. Previous studies have been largely unsuccessful in utilizing AR as the sole drug target for systemic targeted treatment in TNBC. However, there is a lack of other commonly used drug target biomarkers in the treatment of this disease, as well. Thus, analyzing the clinical benefit rate (CBR) within clinical trials that use combination therapy can prove to be imperative to the progression of improving treatment options and prognoses. Breast cancer is one of the most common cancers in women, yet metastatic breast cancer proves to be the least curable. Specifically, TNBC, devoid of commonly targeted receptors, is an aggressive type that is difficult to treat. Additionally, breast-cancer-related deaths are largely related to metastasis . The curGiven CDK4/6’s potent activity in breast cancer cells, researchers aimed to inhibit this activity in conjunction with AR inhibitors . If thesCYP17 lyase inhibitors have been studied extensively in the context of prostate cancer. Many studies have sought its effects specifically in castration-resistant prostate cancer to improve overall survival ,7,8. NewSimilarly, most AR-targeted drugs are approved in the context of prostate cancer, but are still being studied extensively in the context of breast cancer . A combiTNBC is the deadliest subtype of breast cancer comprising 10–20% of all and characterized by its metastatic phenotype . The majThe androgen receptor is a novel target in TNBC. “Luminar AR” (LR) is a subtype of TNBC with a better prognosis but resistance to neoadjuvant chemotherapy. Seminal studies by Lehman et al., 2011, defined four molecular subtypes of TNBC based on their transcriptional profiles: two basal subtypes (BL1 and BL2), a mesenchymal (M) subtype lacking immune cells, and a luminal androgen receptor subtype (LAR) that is enriched in AR expression and follows its transcription program .The initial results from clinical trials indicated a potential clinical benefit for targeting the AR pathway in breast cancer ,16. RetrAR is expressed in normal breast tissue and decreases to 10–50% expression in TNBC ,19. In pAR can also be utilized as a target and possibly be co-targeted with the other signaling molecules it often interacts with, such as CDK4/6, CYP17 lyase, and the PI3K/AKT signaling pathway, as seen in AR is activated by androgens such as dihydrotestosterone (DHT), while its inactive form is bound to heat shock proteins . AR captAR alone has thus proven unsuccessful in acting as a target for treatment. Synthetic steroidal androgens have caused unwanted adverse effects in treatment. The most effective treatment plan includes agents that target AR combined with signal transduction inhibitors . This isClinical trials are currently working on determining the safety and efficacy of combination therapy, including CDK4/6 inhibitors , with anti-androgens such as bicalutamide and with selective AR modulators such as enobosarm. The objective of these studies is to understand the relationship between CDK4/6 and AR and why co-targeting these two important biomarkers can improve CBRs among patients. CDK4/6 inhibitors act to prevent the cell cycle from progressing from the G1 to the S phase . AR’s roAbemaciclib alone has been approved for the treatment of hormone receptor-positive (HR+) advanced/metastatic breast cancer. Compared to the other noted CDK4/6 inhibitors, abemaciclib shows increased potency and specificity for CDK4 based on previous studies . This drBicalutamide trials combined with ribociclib are currently ongoing. Clinical trial NCT03090165, summarized in Enzalutamide has shown greater affinity for the AR target as compared to bicalutamide. In trials of castration-resistant prostate cancer patients, enzalutamide reduced the risk of progression or mortality by 76% compared to bicalutamide . StudiesBicalutamide has shown efficacy alone in treating AR+ BC with a median PFS of 12 weeks . In prevCDK4/6 inhibition is also being utilized in combination with selective AR agonists such as enobosarm in AR+ ER+ HER2- BC. Given that the presence of ER correlates with an upregulation of AR’s tumor suppressor function, trial NCT05065411  will tarAndrogen receptor signaling has been targeted by directly inhibiting AR, or by decreasing the adrenal and tumoral synthesis of androgens. Androgen production is highly dependent on the enzymatic activity of CYP17, which has both 17α-hydroxylase and 17,20-lyase activity . As the Recent phase I and II clinical trials have studied the effects of seviteronel and orteronel in both HR-sensitive breast cancers and TNBCs. Following in vitro studies showing that seviteronel was able to inhibit AR+ TNBC growth on soft agar , the CLAAn early phase 1b clinical trial showed that orteronel was well tolerated in postmenopausal women with HR+ metastatic breast cancer . Of a coSeviteronel has also been shown to work in unison with enzalutamide to radiosensitize AR-positive TNBC tumors more efficiently than when administered individually . This isOther clinical trials are targeting the PI3K/AKT/mechanistic target of the rapamycin (mTOR) pathway via drugs such as alpelisib, taselisib, and ipatasertib in combination with AR inhibitors, such as enzalutamide. The PI3K/AKT/mTOR pathway—a central pathway regulating cell growth and proliferation—is associated with endocrine resistance in breast cancers , and is Following the promising preclinical results, a recent phase Ib/II clinical trial investigated the safety and efficacy of enzalutamide in combination with taselisib, a PI3K inhibitor . In phasThe DESTINY-Breast04 trial used trastuzumab deruxtecan in patients with HER-2-low TNBC tumors. HER-2-low was defined as a 1+ score on immunohistochemical analysis (IHC) or 2+ IHC with a negative in situ hybridization result . The ASCSome TNBC cells may not be completely HER2 negative, rather, they have extremely low HER2 level expression . In factAR and current co-targets have been thoroughly explored throughout this paper, highlighting potential treatment options for patients with AR+ TNBC. Thus far, major co-targets include CDK4/6, CYP17 lyase, and the PI3K/AKT pathway. There are many ongoing clinical trials targeting both AR along with these biomarkers and determining the efficacy of treatment, measured in CBR and PFS, as well as the tolerance of combination therapy, measured in MTD.Future studies can continue the trend of utilizing prostate cancer treatment targets in the study of breast cancer, specifically TNBC. These biomarkers can be co-targeted with AR to improve the effectiveness of the treatment. One possible co-target is the MEK/ERK pathway. Prostate cancer studies have already demonstrated that the IgG1 heavy chain is elevated and when knocked down, there is a downstream inhibition of the MEK/ERK/c-myc pathway . This inThe current clinical trials highlighted here, along with these new potential co-targets and treatment options have exceptional potential and may prove to change the distressing prognosis TNBC patients currently live with.


Patients were evaluated for recurrent instability, revision surgery, and other complications at a mean follow-up period of 64 ± 7.4 months. In addition, Tegner Activity Scale, Lysholm Score, and IKDC-subjective Score were evaluated. Statistical comparison between the two groups was performed utilizing Fisher’s exact test and Student’s t-test. Results: Mean age (29.3 vs. 26.6 years) and preoperative Tegner Score (6.4 vs. 5.9) were comparable between both groups. At 64 ± 7.4 months after ACLR, six cases of recurrent instability were reported in the AccRehab group (18%) in comparison to three cases (9%) in the ConRehab group (p = n.s.). There was no significant difference regarding revision surgery and further complications. Furthermore, no significant difference was found between both groups regarding Tegner (5.5 ± 1.9 vs. 5.5 ± 1.2), Lysholm (93.6 ± 6.3 vs. 89.3 ± 10.7), and IKDC score (89.7 ± 7.9 vs. 86.7 ± 12.1). Conclusion: No significant disadvantage of an accelerated rehabilitation protocol following ACLR was found in terms of recurrent instability rate, revision surgery, or patient-reported outcome. However, a trend towards a higher reinstability rate was found for an accelerated rehabilitation protocol. Future level one trials evaluating brace-free early weight bearing following ACLR are desirable.Introduction: The Purpose of the present study was to assess the outcome of anterior cruciate ligament reconstruction (ACLR) with an accelerated rehabilitation protocol and to compare it to a conservative rehabilitation protocol. It was hypothesized that an accelerated rehabilitation protocol, including brace-free early weight bearing, would result in a higher rate of recurrent instability and revision surgery compared to a conservative rehabilitation protocol. Methods: From 2016 to 2017, two different rehabilitation protocols for isolated ACLR were used at a high-volume knee surgery center. A total of 65 consecutive patients with isolated hamstring ACLR, of whom  Despite advances in the surgical treatment of anterior cruciate ligament (ACL) injuries, recurrent instability following ACL reconstruction (ACLR) occurs in 5% to 20% of patients, with younger and physically active patients at an even higher risk of recurrent instability ,2,3. WhiDifferent phases of rehabilitation following ACLR have been defined ,9,10. DuHowever, a variety of different rehabilitation protocols are currently used by knee surgeons following ACLR ,18. AccoTherefore, the purpose of the present study was to compare the long-term outcome following isolated ACLR with a hamstring tendon autograft, in dependence of the rehabilitation protocol. It was hypothesized that an accelerated rehabilitation protocol including brace-free early weight bearing would result in a higher rate of recurrent instability and revision surgery compared to a conservative rehabilitation protocol.Promising results of accelerated rehabilitation led to the implementation of an accelerated rehabilitation protocol for ACLR. During 2016 and 2017, two different rehabilitation protocols were used at the study center for patients undergoing isolated ACLR with an ipsilateral four-strand hamstring tendon graft. In the present study, consecutive patients between 18 and 60 years of age with isolated ACLR with hamstring tendon autograft in 2016 and 2017 were identified from the study center’s clinical database and included in the study if full consent for the follow-up interview was obtained. Patients with concomitant lesions such as meniscal tears and collateral ligament injuries that would alter the postoperative rehabilitation protocol were excluded. Similarly, those patients with previous knee injuries in both the affected and contralateral knee were excluded. Furthermore, meniscal repair or additional procedures such as lateral extra-articular tenodesis resulted in exclusion from the study.All surgeries had been performed under general anesthesia, with single-shot perioperative antibiotic prophylaxis, by a board-certified surgeon with expertise in ACLR (at least >50 ACLR per year). Anatomic single-bundle ACLR was performed using a four-strand hamstring tendon (semitendinosus) autograft with a femoral fixed-loop suture button fixation and tibial hybrid fixation  . DebrideA co-contraction routine of the quadriceps and hamstrings was practiced from the first day after surgery. Five days after surgery, a brace-free rehabilitation protocol was initiated, including non-weight bearing exercises without limitation in range of motion, full weight bearing was allowed, and progressive training using closed-chain knee exercises was started. Starting four weeks postoperatively, proprioceptive exercises were performed, including dynamic stability training. Running on a level surface was allowed after six weeks, and participation in pivoting sports was allowed after at least eight months and upon successful completion of a return-to-sports test.After four days of knee immobilization in full extension, patients were instructed to wear a hinged hard frame brace for a total of six weeks with a range of motion limited to 90° of flexion and full extension. After two weeks of partial weight bearing with 20 kg, patients were instructed to slowly increase weight bearing with the goal of achieving full weight bearing at six weeks postoperatively. Progressive resistance training using closed-chain kinetic exercises, followed by proprioceptive exercises, was started at six weeks. Straight-line running was allowed at twelve weeks. Pivoting and competitive sports were allowed after at least eight months and upon successful completion of a return-to-sports test. The differences between the two rehabilitation protocols are described in Informed consent to participate in the study was obtained from each patient, and epidemiologic data, as well as surgical measures and incidents during inpatient care, were recorded from the database. Patients were interviewed at a minimum follow-up of five years after surgery. Revision surgery, episodes of giving ways, and subjective instability were recorded. Patients were asked about the occurrence of other complications like joint infection, limited range of motion, or thrombotic events. If a complication occurred, the time after primary ACLR at which the complication occurred was recorded. In addition, the Tegner  activityn = 164 was calculated to obtain a power of 80% at the p = 0.05 significance level to detect a large effect size when utilizing Fisher’s exact test, based on potential differences in the rate of recurrent instability, as supported by the literature [An a priori power analysis was performed using G*Power  . A totalterature ,26.t-test, while non-normally distributed data were compared using the Mann–Whitney test. Frequencies were compared by using Fisher’s exact test. A p-value < 0.05 was considered significant.Statistical analysis was performed using PRISM . Results are presented as means and standard deviations (SD). Criteria for normal distribution of numerical data were checked using histograms and the Shapiro–Wilk test. Normally distributed data were compared using Student’s n = 33 had been treated with an accelerated (AccRehab), and n = 32 with a conservative rehabilitation protocol (ConRehab), agreed to participate in the follow-up examination. No significant difference regarding epidemiologic data was found between the two groups at the time of the initial operation . New trauma during motion was described in all three cases in the ConRehab group and in five of six cases in the AccRehab group. Recurrent instability occurred at 16, 22, and 47 months postoperatively in the ConRehab group and at 9, 10, 12, 12, 20, and 26 months postoperatively in the AccRehab group. Treatment of the recurrent instability was by revision ACLR in two of three cases in the ConRehab group and five of six cases in the AccRehab group.At a mean follow-up period of 64 ± 7.4 months, recurrent instability following ACLR had occurred in 3/32 cases (9%) in the ConRehab group and in 6/33 cases (18%) in the AccRehab group (p = n.s.). Other complications included deep vein thrombosis treated with pharmacologic anticoagulation in one patient in the ConRehab group, in comparison to one contralateral ACL injury and one secondary medial meniscus lesion in the AccRehab group.Arthroscopic arthrolysis for a limited range of motion was performed in four patients in the ConRehab group, whereas no case of arthrofibrosis was observed in the accelerated group , Lysholm (93.6 ± 6.3 vs. 89.3 ± 10.7) and IKDC subjective scores (89.7 ± 7.9 vs. 86.7 ± 12.1). Furthermore, there was no significant difference in patient satisfaction (8.5 ± 1.2 vs. 8.1 ± 2.5). The functional outcome is shown in The most important finding of the present study was that an accelerated rehabilitation protocol with brace-free, early weight bearing after hamstring autograft ACLR, did not result in a significant difference in terms of recurrent instability, revision surgery, and patient-reported outcome compared to a conservative rehabilitation protocol. However, recurrent instability occurred in 18% (6/33) of the AccRehab group in comparison to 9% (3/32) in the ConRehab group, demanding a critical appraisal.The aim of accelerated rehabilitation protocols for ACLR is to achieve early independent daily living with earlier return to work and physical activity without increasing the risk of insufficient graft healing and recurrent instability. However, on the one hand, there is a paucity of studies comparing current rehabilitation protocols for ACLR; on the other hand, there is a variety of accelerated rehabilitation protocols with heterogeneity in the available scientific data ,28.n = 164 required, n = 65 achieved). Therfore, the trend towards increased recurrent instability in the AccRehab groupd might have resulted erroneously non-significant in the present study. However, future trials with larger sample sizes on this issue are desirable.The failure rate of isolated ACLR is typically reported to range from 5 to 10% ,29. A syA hard frame brace is used in the majority of rehabilitation protocols following ACLR to reduce strain on the reconstructed ligament . HoweverA limited range of motion has been reported in 4 to 38% of patients after knee ligament surgery . It has PROMS after ACLR with an accelerated  vs. non-accelerated rehabilitation protocol  was evaluated in a recent randomized controlled trial (RCT). The authors found no significant difference in subjective IKDC score, range of motion, and peak quadriceps isometric strength at 12 and 24 weeks . In anotThe results of the present study are of clinical relevance, considering that ACL reconstruction is the most frequent ligament surgery in Western countries, leading to socioeconomically favorable results, especially in young, physically active patients ,42,43. OHowever, besides the rehabilitation protocol, several factors should be considered, possibly influencing the postoperative outcome and reinstability rate following ACLR. Body mass index (BMI) was shown to be a major influence factor, with obese patients resulting in inferior clinical outcomes after ACL reconstruction in comparison to non-obese patients . FurtherSeveral limitations should be considered when transferring the results of the present study into clinical practice. First and foremost, the inclusion of patients was performed retrospectively, which makes the inclusion process prone to selection bias. However, the comparability of baseline epidemiological parameters and selection from a single center in the same time frame minimizes selection bias. Unfortunately, the results of this study are underpowered regarding the primary outcome (reinstability rate) of the study. Therefore, it is possible that a true difference between the groups was not significant due to the small sample size. Therefore, it is desirable that further clinical studies investigate accelerated rehabilitation protocols on prospective clinical studies with larger sample sizes.It has previously been hypothesized that telephone assessment of PROMs may influence the data obtained with a possible bias towards higher values . HoweverNo significant disadvantage of an accelerated rehabilitation protocol following ACLR was found in terms of recurrent instability rate, revision surgery, or patient-reported outcome in the present study. However, a tendency towards a higher rate of recurrent instability when utilizing an accelerated rehabilitation protocol indicates that level one trials evaluating brace-free early weight bearing following ACLR are desirable.


This systematic review offers an overview on clinical and technical aspects of augmented reality (AR) applications in orthopedic and maxillofacial oncological surgery. The review also provides a summary of the included articles with objectives and major findings for both specialties. The search was conducted on PubMed/Medline and Scopus databases and returned on 31 May 2023. All articles of the last 10 years found by keywords augmented reality, mixed reality, maxillofacial oncology and orthopedic oncology were considered in this study. For orthopedic oncology, a total of 93 articles were found and only 9 articles were selected following the defined inclusion criteria. These articles were subclassified further based on study type, AR display type, registration/tracking modality and involved anatomical region. Similarly, out of 958 articles on maxillofacial oncology, 27 articles were selected for this review and categorized further in the same manner. The main outcomes reported for both specialties are related to registration error  and surgical accuracy  obtained under AR navigation. However, meta-analysis on these outcomes was not possible due to data heterogenicity. Despite having certain limitations related to the still immature technology, we believe that AR is a viable tool to be used in oncological surgeries of orthopedic and maxillofacial field, especially if it is integrated with an external navigation system to improve accuracy. It is emphasized further to conduct more research and pre-clinical testing before the wide adoption of AR in clinical settings. Augmented reality (AR) is a technology that allows the fusion of digital content into the real environment. The achieved augmented continuum is a virtual world in which virtual objects are overlaid on real elements, in the surrounding actual environment .The first AR system using a Head Mounted Display (HMD) was developed by Sutherland in 1968 . Since iWhen applied to surgery, the AR allows to improve the user’s perceptual and comprehensive ability by projecting three-dimensional underlying anatomy directly onto the user’s retina (via HMDs) or on a display screen.AR in surgery has enormous potential to help the surgeon in identifying tumor locations, delineating the planned dissection planes, and reducing the risk of injury to invisible structures. Therefore, using AR in the operating room (OR) could be helpful in performing surgical tasks in a more accurate way. HMDs are particularly beneficial for AR surgical applications since they intrinsically provide the surgeon with an egocentric viewpoint, and offer improved ergonomics if compared to traditional computer-assisted surgical systems. This allows surgeons to concentrate on the task at hand without having to turn their heads away from the surgical field to constantly look at imaging monitor. The most ambitious goal in surgery is to use AR for intraoperative navigation. This involves taking data from preoperative imaging and using anatomical anchors in the operating field to register the two representations in real time.Registration is an important step in computer-assisted surgical navigation in order to correlate the virtual content and the real surgical scene. In this context, the registration error can be defined as; the measurement of how much the virtual objects displayed in AR appear incorrectly positioned relative to the real environment.For virtual-to-real surgical scene registration, AR systems typically use a camera coupled to a device marker; such as QR code, anchored to the patient (marker-based registration). Another option is marker-less registration which includes a combination of location data , inertial measurement unit (IMU) data, and computer vision to track image features such as scene depth, the object surface, and object edges .The core of the registration modality is tracking, which means to determine and follow the position and orientation of an object with respect to some reference coordinate system over time.Over the past decade, with the advent of multimodal and high-detailed 4D medical imaging , numerouOrthopedic and Maxillofacial surgeries have been pioneers in the use of AR in a surgical setting .These two surgeries may represent very promising fields for the future clinical implementation of AR, since they are based on bony hard tissues which make it easier to have fixed references, i.e., bony structures, to be used for ensuring an accurate virtual-to-real scene registration between preoperative  and intraoperative  views.in vitro study on sawbone models for employing AR during total knee arthroplasty and concluded that the system provided accurate measurements for tibial bone resection. Consequently, in 2021, the same authors, formulated prospective cohort study on 72 patients. They emphasized that AR-assisted navigation to resect distal femur is more precise than the conventional method  method was used to construct the suitable research question: “Can augmented reality be considered a beneficial tool in orthopedic and maxillofacial oncological fields in achieving surgical accuracy?”This review addresses this question by focusing on both clinical and technical aspects of AR in these two surgical disciplines, as well as on reporting current limitations and benefits.Due to the qualitative and mixed-method nature of the included articles and the heterogeneity of the data, the term “evaluation” was left intentionally broad.We performed the study selection based on the following inclusion criteria: studies on augmented reality applications in either orthopedic or maxillofacial field, only focused on oncology; studies reporting applications on different targets . All articles with either quantifiable or qualitative outcomes on augmented reality confined to both specialties with case reports were included.Exclusion criteria were the following: articles “not relevant” ; articles with language of publication other than English; theses; conference papers; editorials; book chapters; review articles .All the search articles available till 31 May 2023 were screened by title first and then abstract.® . All articles meeting the inclusion criteria were read carefully and stratified following two parallel perspectives: a clinical one, i.e., focusing on the specific surgical application, the type of study , the anatomical region of interest, the virtual information provided to surgeon, and a technical one, i.e., the registration/tracking modality the type of AR display, the achieved registration and surgical accuracy).The authors, date of publication, study design, and data from the eligible articles were tabulated in Microsoft ExcelKey findings of each article were also stated in given tables in the Results section for both orthopedic and maxillofacial specialties, and also depicted in bar histograms. However, meta-analysis could not be performed due to heterogeneity of literature. All these findings were validated by a second independent investigator to ensure the correct data acquisition and selection of the appropriate relevant literature.Due to the fact that the included study designs exhibited a significant level of variability, as is often observed in the case of new technologies, they are developed individually with distinct features. Consequently, conventional approaches for evaluating the risk of bias were not suitable for use in this context. The authors generally evaluated and assessed the risk of bias to be low or negligible for data description, but it could be high for the analysis of the effectiveness of approaches used in these studies. Furthermore, none of the articles included in the review refers to a specific methodological protocol.The initial search of the PubMed/Medline and Scopus databases was completed on 31 May 2023, and all available articles were scrutinized using the above-described criteria.In the following paragraphs, an analytic overview of the selected papers and their classification were presented for both orthopedic oncology and maxillofacial oncology.n = 27), review articles/book chapters/editorials/conference papers (n = 22), non-English articles (n = 1).As depicted in flowchart , the datn = 395), review articles/book chapters/editorials/conference papers (n = 286), non-English language (n = 30).Similarly, for CMF oncology, 951 articles were found through PubMed/Medline and Scopus search and 7 from manual search. Out of total 958, 738 articles remained after removal of duplicates. Based on above defined criteria, 27 publications were included and others were eliminated for the following reasons: “not-relevant”  or both on phantoms and prospectively on patients (n = 3). Three studies were conducted on animal cadavers, whereas, two studies directly employed AR on patients in OR. In the context of AR display and registration/tracking modality, five studies used screen-based display and eight studies opted for a marker-based registration/tracking modality. Regarding the anatomical region of interest, pelvis is the most involved anatomical region in the selected studies (n = 4).For orthopedic oncology , four stThe main outcomes reported in the included articles referred to the registration error of the AR systems, the AR-guided surgical accuracy in performing tumor resection compared to preoperative planning and/or to standard procedures , the placement error in positioning surgical guides or patient-specific implant under AR assistance.Despite the fact that the application of AR in the field of orthopedic oncology has been relatively limited compared to maxillofacial oncology to date, the included studies demonstrate the potential future significance of AR technology in this surgical field.n = 14) the proposed AR systems were tested on phantoms, and 8 on patients only. Five researcher groups carried out pre-clinical research on phantoms before using AR on patients. The marker-less registration approach (n = 14) and wearable AR displays, i.e., HMDs (n = 14), were utilized in the majority of the research studies. These two investigating factors made up 52% of the total studies. Mandible, being the most involved anatomical area of interest for AR implementation, comprises of 52% of total studied areas (n = 14).In the field of maxillofacial oncology , in the The following are the primary findings that are covered in the articles: the registration error, the AR-guided surgical accuracy in performing tumor resection or flap harvesting compared to preoperative planning, and the AR-guided surgical accuracy compared to the more conventional use of 3D printed cutting guides. It is interesting to mention that, some studies in maxillofacial oncology have also incorporated external tracking navigation systems into augmented reality to improve accuracy and spatial relationships. The articles included have shown the importance of AR and its future perspectives in this field. Nevertheless, the majority of the studies underlined the need for additional research before clinical application.In the set of orthopedic oncology articles, all studies utilized marker for registration except 1 , which oOut of nine articles, only three randomized controlled trial studies (RCT) were reported . Mean AR-assisted resection was less than 2 mm as compared to conventional approach  which had mean resection error more than 2.5 mm in these studies .Included studies of orthopedic oncology also reported two cases, both achieved intended outcomes using AR without any complications . MoreoveRegarding usability, only one study conducted a questionnaire survey with patients and surgeons, and the results turned out to be satisfactory . ErgonomThe primary outcomes of the included articles in orthopedic segment demonstrated the utility of AR and showed even better results than conventional procedures in some cases. However, some of the limitations of the orthopedic segment is that few articles discuss the usability and ergonomics of this technology. Only three out of nine articles are randomized controlled trials (RCTs), making it difficult to derive a statistical comparison value from the current studies. Furthermore, only four articles provided surgical accuracy  and onlyOver all, with consideration of sufficient accuracy achieved in terms of registration error and surgical accuracy during surgical simulations, it can be said that AR is beneficial and helpful in orthopedic oncological surgeries. However, due to limited literature in orthopedic oncology till date, further testing is recommended.In maxillofacial oncology, out of eight marker-based studies, only two reported AR regisration error in terms of fiducial registration error (<1 mm) . Marker-Marker-based AR surgical accuracy was reported in five articles and it was demonstrated in terms of deviation from pre-planned surgical resection. Mean surgical resection error was measured to be less than 3 mm among these articles . ConversTwo studies employed both marker-less and marker-based registration, the latter being used mainly in the case of lack of surface features easily recognizable  or to inTwo of the three case reports on maxillofacial oncological surgery were qualitative, while one case of fibrous histiocytoma had quantified findings with an overall mean discrepancy of 2.77 ± 1.29 mm using AR . AccordiA total of seven studies have examined the utilization and precision of AR in the context of flap harvesting for mandibular restoration . Among tIt is interesting to outline that five of the studies utilized external navigator system with mixed reality. Four of them incorporated external navigation into mixed reality to enhance accuracy and spatial relationships  and one Feasibility and usability studies were conducted in two articles in maxillofacial oncology. In one study, the standard ISO-9241/110 feasibility questionnaire based on the 5-point Likert Scale was conducted on both medical staff and AR experts, and overall feedback turned out to be positive . In anotIn addition, AR guided simulations scored higher as compared to virtual unguided resections in mental demand, performance, effort and frustration in preclinical study conducted on phantoms by Some clinicians find the AR system simple to learn and use, which improves their decision-making skills , whereasDespite of the fact that a few studies did not achieve sufficient accuracy in terms of registration error and surgical accuracy in maxillofacial oncology, the overall outcomes seem to have a positive impact of AR in maxillofacial oncological surgeries. For instance, according to Similar to orthopedic oncology, maxillofacial oncology section also lacks in many aspects. Feasibility questionnaire survey and ergonomics are discussed in only few articles. The different methods of evaluation in articles limited the ability to provide quantifiable results. Additionally, only 10 out of 27 articles reported on registration error and only 14 reported on surgical accuracy. Conversely, further research is emphasized pre-clinically before implementing AR in operating rooms.We should advocate for the development of a technique that is uniform and consistent in order to investigate this new technology and make it possible to conduct meta-analyses for future investigations. This stage is crucial for gathering data to support the use of AR in oncological procedures in both disciplines. Additionally, we stress the importance of including external navigation in AR in future experiments in order to enhance the precision and depth perception of this infant, yet useful technology. Moreover, through the use of standardized questionnaires, SUS, and a 5-point Likert scale, feasibility and ergonomics should be evaluated.We observed from the collected papers that an important aspect of AR implementation is three-dimensional printing (3D printing), also referred to as rapid prototyping, which is typically used for obtaining pre-operative patient-specific phantoms replicating the anatomical structures of interest. These phantoms are typically used in the papers to perform the surgical task preoperatively under AR guidance, as well as to evaluate both the registration error and surgical accuracy . When usDespite the fact that AR is a growing technology, it is not without limitations and complications. Surgeons should be well aware of the limitations of augmented reality in surgery, including technical challenges, limited field of view which limits the amount of virtual content available to the user, high implementation costs and limited user experience. In addition, they should consider how these limitations may impact the accuracy and efficacy of AR systems, as well as the surgical outcomes. Before incorporating AR into clinical practice, it is essential to execute a comprehensive analysis of its viability and benefits.For instance, the viewing distance and angle of commercially available HMDs, such as HoloLens 2, are not optimized for use in surgery since the focus distance is suboptimal for medical procedures that are typically carried out at arm’s length and with the head bowed to observe the operative field .https://www.augmedics.com/), and the VOSTARS system, still under investigation (https://www.vostars.eu/). VOSTARS is promising new wearable AR system designed as a hybrid Optical-See-Through (OST)/Video-See-Through (VST) HMD capable to offer a highly advanced navigation tool for maxillofacial surgery and other open surgeries. An early prototype of the VOSTARS system , which can result in technical issues such as system failure, calibration errors, and latency. These obstacles may limit the accuracy of AR systems and result in surgical complications .In addition, surgeons using AR-HMDs must contend with the limited field of view, restricted binocular field and projection size .Virtual-to-real scene registration with HMDs is another major issue when using AR for surgical guidance and simulation, resulting in inaccurate identification of the deep anatomical structure in question. Due to the fact that these display devices were not devised for medical purposes, their technical characteristics are less suited for surgical procedures .Surely, a marker-less registration, i.e., without the use of fiducial markers or trackers anchored to the patient, is highly preferable in surgery, however it is not always feasible for certain surgeries. As also emerged from our analysis, in maxillofacial oncological surgery a marker-less based approach seems to be more viable since the edges of anatomical parts  are more accessible and trackable during surgery; conversely, in orthopedic field the intraoperative recognition of bone edges may be more difficult.During surgery, a surgeon using an AR headset may endure discomfort, weariness, eye strains, and headache. Furthermore, it is possible that the surgeon’s ability to focus on the surgical operation field might get impaired due to the visualization of augmented information. However, in a study conducted on simulation sickness, in 2018, authors showed that out of 142 HMD’s users from various fields, only few experienced mild discomfort .Because this technology is high priced and requires significant investment to initiate and implement in hospitals, it is not widely accessible in all medical settings. Consequently, the data associated with augmented reality in the surgical field are rather preliminary and require further testing and analysis.The AR application has a steep and costly learning curve. It requires expertise, and most surgeons are unfamiliar with AR utilization. So they must collaborate with biomedical engineers to implement this technology in the operating room. Personnel must endure time-consuming hands-on training in order to implement AR in hospital surgical setups.When information needs to be electronically distributed to several departments in order to make patient-specific tools for AR, patient data privacy is another concern that must be addressed. When dealing with sensitive information pertaining to patients, certain national regulations should be followed in order to protect and secure both their safety and their privacy.Augmented reality is being recognized as a promising application for enhancing the outcomes and standard of care for orthopedic and maxillofacial oncology patients. Especially in complex oncological cases that require a strategic planning for execution and comprehension, it is emphasized that surgeons should consider using augmented reality where applicable, in combination with 3D printing. However, implementation of AR and its tools in surgical cases and healthcare has certain shortcomings which can be improved with future advancement in technology. Depending on the complexity of cases, process from procuring CT scan/MRI images to 3D printing of pre-surgical phantoms and developing a patient-specific AR application take days, sometimes even months including preoperative surgical planning and simulation training. Surgeons and biomedical engineers must work together to refine and successfully execute the procedure. Therefore, as future perspective, AR cannot be used in emergency cases and can only be advised in elective surgeries. Despite these facts, we continue to believe that AR technology has the potential to revolutionize the conventional methods of oncological surgical procedures and overcome all of these limitations. Augmented reality is expected to help in better understanding of tumor anatomy and in plan the resection accordingly. This will contribute to improve the outcomes and standard of care by limiting the recurrence rate while attaining the desired surgical margins with accuracy.Currently, augmented reality is one of the most innovative technology in the field of surgery, particularly orthopedic and maxillofacial surgery. Due to its real-time visualization of preoperative images and planning directly on the patient, AR can be particularly beneficial for oncological surgeries in both fields to achieve the desired surgical accuracy. In oncological surgery, the AR allows to overcome some limitations of conventional computer-assisted surgical navigation, such as the surgeon’s attention shift from the operative field to view the navigation monitor, as well as to avoid the lead-time in manufacturing 3D-printed cutting guides. Indeed, AR should be used as a complementary tool to other computer-assisted technologies, as suggested by our literature review: particularly for maxillofacial oncology, surgeons have begun to incorporate external navigation systems into AR to track the surgical probe or instruments, to further improve accuracy and spatial relationships.Even though AR technology is still in its infancy and has certain limitations, the current outcomes of its application in both disciplines are promising to support its clinical use. Certain concerning aspects still remain, related to image-to-patient registration and surgical accuracy. In the present review, we attempted to identify a range of registration error and surgical accuracy based on results from both surgical domains. Although it is difficult to derive general ranges due to the involvement of various anatomical regions and different complexity for each domain it can be observed that AR resection error exhibited greater accuracy compared to conventional un-guided resection, hence successfully attaining the desired goals without any associated complications. Additionally, in some studies the AR navigation showed comparable accuracy with pre-planned virtual cutting planes and with customized cutting guides.We believe that the still limiting technical aspects on registration and surgical accuracy can be improved and overcome with further development in hardware and software used for AR. For that, industry-academic partnerships are essential to advance the technology, in conjunction with clinical studies to assess its benefits and role in the clinical practice.In conclusion, although AR is seen to have the capacity to enhance surgical efficiency and ensure patient’s safety, further search needs to be done pre-clinically in order to improve its accuracy and before achieving its wide adoption in clinical settings.


Mechanistically, acK609-AR was recruited to the AR and ACK1/TNK2 enhancers, up-regulating their transcription. ACK1 kinase–mediated AR Y267 phosphorylation was a prerequisite for AR K609 acetylation, which spawned positive feedback loops at both the transcriptional and posttranslational level that regenerated and sustained high AR and ACK1 expression. Consistent with these findings, oral and subcutaneous treatment with ACK1 small-molecule inhibitor, (R)-9b, not only curbed AR Y267 phosphorylation and subsequent K609 acetylation but also compromised enzalutamide-resistant CRPC xenograft tumor growth in mice. Overall, these data uncover chronological modification events in AR that allows prostate cancer to evolve through progressive stages to reach the resilient recurrent CRPC stage, opening up a therapeutic vulnerability.Resistance to second-generation androgen receptor (AR) antagonists such as enzalutamide is an inevitable consequence in patients with castration-resistant prostate cancer (CRPC). There are no effective therapeutic options for this recurrent disease. The expression of truncated AR variant 7 (AR-V7) has been suggested to be one mechanism of resistance; however, its low frequency in patients with CRPC does not explain the almost universal acquisition of resistance. We noted that the ability of AR to translocate to nucleus in an enzalutamide-rich environment opens up the possibility of a posttranslational modification in AR that is refractory to enzalutamide binding. Chemical proteomics in enzalutamide-resistant CRPC cells revealed acetylation at Lys AR enhancer has also been reported wherein histone Y88 phosphorylation marks were deposited 94, 114, and 132 kb upstream of the start site, causing increased AR and AR-V7 expression in CRPC (Therapies directed against the androgen receptor (AR) signaling axis are the mainstay of treatment regime for patients with early- or later-stage metastatic prostate cancer . Often,  in CRPC . AlthougR)-9b would escalate the antineoplastic response in recurrent CRPC.Here, we report a modification in AR K609 acetylation that depends on AR Y267 phosphorylation exerted by nonreceptor tyrosine kinase Activated Cdc42-associated kinase 1 (ACK1). This “dual- modified AR” regulates the expression of AR and ACK1 that phosphorylates AR at Y267, thereby creating a self-sustaining circuit that maintains high expression of AR and ACK1. In this study, we sought to unravel the mechanistic details of successive interdependent AR modification events that impart resistance to AR antagonists and further explored whether targeting the ACK1-AR nexus with small-molecule inhibitor (2H), allowing bound proteins to be captured by streptavidin- coated Sepharose beads (R)-9b, were used as a negative control -9b, which inhibits AR K609 acetylation, followed by immunohistochemical (IHC) staining using acK609-AR antibody. As displayed in R)-9b–treated cells. Cells stained with just primary or secondary antibody were used as controls to negate background staining (K609 in the second zinc finger of the DNA binding domain (DBD)  is consestaining . The expstaining , which istaining . The acestaining .To examine whether AR K609 acetylation directly correlated with AR antagonist treatment, we treated C4–2B and LNCaP cells with enzalutamide and abiraterone, which revealed increased acK609-AR . We examAR gene locus by depositing histone H4 Tyr88 marks in the AR enhancer, a crucial step for efficient transcription of AR and AR-V7 in CRPCs (R)-9b, which exhibits both good potency  and selectivity (R)-9b on the abundance of acK609-AR, we subjected VCaP, LNCaP, and C4–2B cells to (R)-9b treatment. A decrease in acK609-AR was observed upon (R)-9b treatment (R)-9b exhibited minimal effect on the total global acetylation in prostate cancer cells -9b–treated enzalutamide-resistant VCaP cells was immunoprecipitated (ChIP) with acK609-AR antibody, followed by next-generation sequencing (ChIP-seq). Peak analysis revealed acK609-AR binding to 727 distinct sites; acK609-AR peaks were primarily annotated in intergenic regions, including enhancers and introns (R)-9b treatment, are shown in R)-9b–treated cells -9b treatment . Ot. OtHSD17AR and ACK1 enhancers (PDCD6IP and NRG3 (R)-9b treatment. This binding was enhanced by the HDAC inhibitor SBHA, which prevented AR K609 deacetylation, and was diminished by the HAT inhibitor E , and NRG3 genomic loci . Compared with AR, the acetylation-deficient K609A-AR mutant exhibited decreased binding at ACK1  mutant of AR (K609A-AR) and transfected the constructs in prostate (PC3 and DU145), breast (MCF7), and human embryonic kidney HEK) 293 cells with low or undetectable AR expression. The K609A-AR constructs exhibited decrease in AR K609 acetylation . We next at ACK1 , B and CACK1 -9b, SBHA, or C646. Both (R)-9b and C646 exerted an inhibitory effect on the AR (PDCD6IP (KLK3 (R)-9b caused down-regulation of the acK609-AR–regulated genes HSD17B11, SIM2, ZRANB3, and ADAMTSL3 , AR-deficient HEK293, PC3, and MCF7 cells were cotransfected with AR, K609A-AR, or K609Q-AR mutants and an ARR2PB-luciferase reporter construct. DHT treatment caused an increase in the luciferase activity of AR; however, luciferase activity of the acetylation-deficient K609A-AR mutant was compromised , the K609 from each monomer is positioned within the minor groove to interact with the phosphate backbone of the response element , A to C.R)-9b, and IP was performed with p300 antibody, followed by immunoblotting with AR. An AR/p300 complex was observed in vehicle-treated samples; however, complex formation was compromised upon (R)-9b treatment -9b and p300 inhibitor C646. In contrast, ACK1 and p300 binding was up-regulated upon treatment with the HDAC1/3 inhibitor SBHA , the mice were randomized and injected with either vehicle [6% Captisol in phosphate-buffered saline (PBS)] or (R)-9b (12 or 20 mg/kg of body weight) five times a week. Tumor measurements were performed twice a week. CRPC tumor growth was compromised upon (R)-9b treatment, and the dose at 12 mg/kg was found to be sufficient to overcome CRPC tumor growth (R)-9b–treated CRPC tumors displayed a reduction in weight, in contrast to the robust tumor growth seen in vehicle-treated mice (R)-9b–treated mice (R)-9b is not toxic.To investigate the effect of inhibition of AR K609 acetylation on CRPC xenograft tumor growth, enzalutamide/abiraterone-resistant C4–2B cells were implanted subcutaneously in castrated male severe combined immunodeficient (SCID) mice. When the tumors became palpable -9b on the host, total RNA was isolated from the prostate and brain of these mice. The brain was chosen because it is known to have AR expression, and it is important to determine whether (R)-9b passes the blood-brain barrier. No marked deviation from normal was observed in prostate or brain AR expression upon (R)-9b treatment -9b treatment caused a marked decrease in pACK1 and ACK1 protein in comparison to the vehicle. This decline was accompanied by a corresponding suppression of pY267-AR and acK609-AR expression (R)-9b has no adverse side effects on the physiological well-being of the mice, as reflected by a lack of change in mice weight or AR abundance in host organs such as the prostate and brain; however, AR expression in CRPC tumors was specifically compromised by (R)-9b. Combined with our previously published findings that show specific activation of ACK1 in prostate tumor cells (R)-9b leads to a loss of acK609-AR, resulting in a comprehensive response against CRPC.To further gauge the effect of -9b can exert its tumor-suppressing effect when administered orally, enzalutamide-resistant VCaP cells were implanted subcutaneously in male SCID mice. When the tumors reached about 100 mm3 in size, the mice were randomized and received vehicle (6% Captisol) or (R)-9b (24 or 48 mg/kg of body weight) by oral gavage five times a week. (R)-9b– treated mice displayed a marked reduction in tumor volume and weight, as opposed to the robust tumor growth observed for vehicle-treated mice (R)-9b at 48 mg/kg, eight mice formed tumors, which were also markedly smaller (R)-9b injection, oral gavage of (R)-9b also caused a marked reduction in AR, KLK3, and TMPRSS2 mRNAs in comparison to the vehicle-treated group (R)-9b also caused a reduction in pY284-ACK1, leading to the loss of pY267-AR and, hence, acK609-AR expression (R)-9b treatment. These data provide evidence of feasibility of oral dosing of (R)-9b, emphasizing the importance of this route and formulation for future clinical trials.To investigate whether -9b–treated C4–2B and VCaP xenograft tumors (R)-9b–treated tumors, which was reflected in a suppression of pY267-AR and acK609-AR staining   . Overall × 10−5) .AR and ACK1.In this report, we uncover a dual-modified AR engendered by cancer cells to maintain high AR and ACK1, overcoming the effect of AR antagonists. Because of successive deposition of these modifications and their interdependence, targeting the earliest event, AR Y267 phosphorylation, using an ACK1 kinase inhibitor not only suppressed AR K609 acetylation but also caused a global shutdown of many critical genes, including 609 acetylation occurs in the vicinity of the distal box region, which enables AR dimerization and DNA interaction (The AR DBD (residues 556 to 623) is conserved in steroid hormone receptors. Lyseraction , indicateraction –36. It wIn the x-ray crystal structure of the AR DBD complexed with the ARE, the major driver of protein-DNA interaction is binding of the AR DNA recognition helix in the major groove of the ARE , 37. Loc267 is located is not available. CBP/P300- interacting transactivator with Glu/Asp- rich C-terminal domain 2 (CITED2) is a transcription cofactor that interacts with several other transcription factors and cofactors and is shown to have a role in multiple cellular processes, including proliferation, apoptosis, differentiation, migration, and autophagy -9b as a desirable therapeutic modality.Other genes that are regulated by acK609-AR include single-minded homolog 2 (SIM2), which is known to be the highly up-regulated gene in prostate cancer , and itsIt has been reported that AR is acetylated at K630/632/633 in the hinge region (between the DNA and ligand-binding domains) by p300 and P300/CBP-associated factor (PCAF), important for the ligand-dependent AR function , 49. HowThe inability of the K609 acetylation–deficient mutant to optimally trigger AR transcription activity using rat probasin (PB) promoter in the presence of DHT suggests that ac-K609 also has a role in canonical AR activation. The androgen-regulated Probasin (PB) promoter region has transcription factor–binding sites for FoxA1, Oct-1, c-jun, and neurofibromatosis-related protein (NF1) . All theRecently, mRNA sequencing of 550 primary and 120 mCRPC samples demonstrated that CBP and p300 mRNA expression is associated with the AR signature . FurtherCoordinated phosphorylation/acetylation modifications are also reported for the regulation of DNA binding by transcription factors, such as STAT proteins, however, with different consequences . AcetylaR)-9b in organoids derived from CRPCs and enzalutamide-resistant PDX tumors, which is a prerequisite for a prospective clinical trial of ACK1 inhibitors.Our study has some limitations. Compared to AR, whether acK609-AR forms a distinct transcription complex is not clear. A common set of motifs recognized by acK609-AR in both enzalutamide-resistant VCaP and LNCaP cells indicates that CRPCs sense loss of AR activity, promoting a distinct transcription program using acK609-AR. How prostate cancer cells summon HATs to acetylate AR in response to AR antagonists is not clear and will be the topic of future investigation. In addition, we have not compared direct AR and acK609-AR DNA binding to chromatin in the absence of its ligand, DHT, which would be indicative of relative binding efficiency in AR antagonist–rich environment. Moreover, we have not yet tested the effect of . For xenograft studies, mice were randomly assigned to groups before initiating the experiment, examined daily, and humanely euthanized at defined study end points. Experimenters were not blinded to group allocation. All animal procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Washington University in St Louis. For other assays, at least two or three independent experiments were performed, each with triplicates, with a representative study shown.K-ac]NCPSCRL and a non-acetyl peptide: TIDKFRRKNCPSCRL. Five mice were immunized four times with acetyl peptides, several weeks apart, and an enzyme-linked immunosorbent assay was performed to determine the relative titer of sera against acetylated and non-acetylated peptides. The mice with the best titer (and least reactivity with non-acetylated peptide) were chosen for fusions. The supernatants of the clones were tested against peptides and immunoblotting of proteins. Two clones were selected, 5B9E4 (#7) and 5B9E5 (#8). The 5B9E4 (#7) clone supernatant was primarily used in this study.The antibody expressing clones were custom-generated by ProMab. Briefly, two AR peptides coupled to immunogenic carrier proteins were synthesized, an acetyl peptide: TIDKFRR[n = 1) were assessed for AR acetylation/phosphorylation and ACK1 phosphorylation. Patients were consented to our IRB-approved genitourinary banking protocol [Human Research Protection Office (HRPO) no.: 201411135] to allow tissue collection. Tissues were fixed and sectioned, and hematoxylin and eosin staining was performed followed by pathologist review to determine the status of samples. IHC staining was performed for the tissue sections at the following concentration: acK609-AR (1:1), pY267-AR (1:50), and pY284-ACK1 (1:300). Pathologist’s (C.W.) scores for intensity of the staining (range: 0 to 4) are shown below the images.Patients with localized prostate cancer, metastatic hormone-sensitive prostate cancer, or mCRPC were recruited for the study. Prostate tumor and adjacent benign tissues from an enzalutamide-treated patient . C4–2B cells (2 × 106) were suspended in 200 μl of PBS with 50% Matrigel (BD Biosciences) and were implanted subcutaneously into the dorsal flank of castrated 6-week-old male SCID C.B17 mice (n = 7 per group). Once the tumors become palpable , mice were injected subcutaneously with (R)-9b resuspended in 6% Captisol (or 6% Captisol in PBS as vehicle) at the concentrations 12 and 20 mg/kg of body weight, five times a week, for 4 to 5 weeks. Tumor volumes were measured twice a week using calipers. Formation of tumors was monitored over a 10-week period. At the end of the study, all mice were humanely euthanized and tumors were extracted and weighed.All animal experiments were performed using the standards for humane care in accordance with the National Institutes of Health (NIH) n = 10 per group) and treated orally with 24 and 48 mg/kg of body weight with (R)-9b in 6% Captisol (or 6% Captisol in PBS as vehicle) five times a week. IHC staining was performed for the tumor sections as described above.For the oral efficacy study, a similar strategy was used except that the VCaP cells were injected in male SCID C.B17 mice . All tests were two sided, and statistical significance was defined at 5% alpha.For the tissue microarray analysis, Kruskal-Wallis tests were performed to examine differences in nuclear pY284-ACK1 and acK609-AR with respect to different progression stages of prostate cancer. Boxplots were used to summarize the intensity distribution at each progression stage. Analysis of variance (ANOVA) was done to examine differences in nuclear expression ofpTyr284-ACK1 and acK609-AR at different tumor stages, and Tukey’s method was performed to adjust for all pairwise comparisons. Correlations were done using Spearman rank-based correlation. All analyses were conducted using GraphPad Prism (GraphPad Software Inc.) and R -9b, can be made available through a material transfer agreement with Moffitt Cancer Center, Tampa, FL.All data associated with this study are present in the paper or the SI information Sci Tranl Med