TRAMP-C2, HEK293T and Jurkat cells were obtained from ATCC. LAPC4 and C4-2B cells source and growth conditions are described earlier13. TRAMP-C2 were grown in DMEM containing 4 mM L-glutamine and 1.5 g/L sodium bicarbonate supplemented with 10 nM dehydroisoandrosterone 90%, 5 μg/ml bovine insulin, 5% FBS and 5% Nu-Serum IV. HEK293T cells and Jurkat cells were cultured in DMEM and RPMI-1640, respectively, supplemented with 10% FBS. All cultures were maintained with 50 units/ml of penicillin/streptomycin (Invitrogen) and cultured in 5% CO2 incubator. All cultures were tested for mycoplasma contamination every 2 months using the PCR Mycoplasma Test Kit I/C (PromoKine). Identities of all cell lines were confirmed by Short Tandem Repeat (STR) Profiling.
Generation of Tnk2 conditional KO mice using Cre-loxP recombination strategy
All animal experiments were performed using the standards for humane care in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mice studies were performed according to IACUC protocols approved in writing by Washington University in St. Louis Department of Comparative Medicine (IACUC protocol nos. 20180247 and 20180259). All mice were co-housed with 3–5 mice per cage and maintained in a controlled pathogen-free/germ-free environment with a temperature of 20–23 °C, 12/12 h light/dark cycle, 50–60% humidity, and food and water provided ad libitum. The Tnk2 gene (NCBI Reference Sequence: NM_016788; Ensembl: ENSMUSG00000022791) is located on Mouse chromosome 16. 16 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 16 (Transcript: ENSMUST00000115124). The 5’ loxP site is located in intron 2 and the 3’ loxP site is inserted in intron 3. This region is followed by the frt-flanked PGK promoter driven SV40-neo gene, located in intron 2. Cre-mediated recombination between these two sites will result in deletion of part of intron 2 and all of exon 3 to generate a stop codon TGG due to splicing of exon 2 with exon 4, causing premature termination (Supplementary Fig. 1b). The targeting construct was electroporated into C57BL/6 (black) embryonic stem (ES) cells. Cells containing the correctly targeted allele were identified by PCR using a forward primer in the genome, outside the region of targeting and reverse primer in the neo gene. Neo and 5’ loxP sites were introduced into the second intron along with a new BamHI site just after 3’ loxP in intron 3. As a result digestion of genomic DNA from G418 resistant clones resulted in the appearance of a 9.8 kb and a 13.5 kb bands corresponding to the wildtype and neo inserted alleles respectively. 23% of the clones were tested positive by PCR and the clones were reconfirmed by southern blotting.
Two embryonic stem cells (ES) clones (BO4 and CO4) each containing a single targeted Ack1 allele were microinjected into albino C57BL/6 (B6) blastocysts. Four mice with 40–75% of chimera were observed. Chimeric males were then mated with B6 albino females to screen for germ line transmission. Two black pups, likely to be floxed for Ack1 or Ack1flx/wt were obtained. Ack1flx/wt mice were bred with EIIa-Cre mice to determine whether loss of Ack1 leads to embryonic lethality. The adenovirus EIIa promoter directs expression of Cre recombinase in preimplantation mouse embryos and in nearly all tissues. No embryonic lethality was seen. We obtained Ack1 heterozygous mice, which were subsequently interbred to obtain homozygous knockout mice (Supplementary Fig. 1c, d). The tail PCR was done to confirm genotypes of the mice. Ack1 KO mice were backcrossed to C57BL/6 for at least 10 generations.
ALT activity was determined in liver lysates from the WT and Ack1 KO mice using the Alanine Transaminase Activity Assay Kit (Abcam), according to the manufacturer’s recommendations. The experiment was done in triplicates and the mean value has been plotted.
Blood serum was collected from the WT and Ack1 KO mice and the ANA assay was done using Mouse anti-nuclear Antibody (IgG) ELISA Kit (CUSABIO, Wuhan, China) according to manufacturer’s protocol.
Histology and staining
Tissues collected from the WT and Ack1 KO mice were fixed in paraformaldehyde and paraffin embedded. 5-μm sections were cut and stained with H&E and Picro-Sirius red. Based on the degree of severity, inflammation and fibrosis were evaluated by pathologist (C.W.).
We used ClusPro34, ACK1 (PDB-entry 4HZR)33 as receptor and CSK (PDB-entry 1K9A)32 as ligand. We generated restraints for docking from the co-crystal structure of insulin receptor kinase (IRK) with peptide (PDB-entry 1IR3)35 by aligning IRK on ACK1 and calculating seven Cα-Cα distances between structurally conserved residues on ACK1 and the position of the substrate tyrosine bound to IRK. We performed multiple parallel docking runs while relaxing restraints from 2.5 Å to 5 Å. Docking solutions were manually inspected. We chose the solution with the most similar backbone geometry around Y18 to that of the substrate peptide observed in PDB-entry 1IR3. We aligned the structure of IRK (PDB-entry: 1IR3) on ACK1 kinase when complexed with CSK to position the ATP and two Mg2+ ions. The complex was analyzed in COOT36 and the side chain chi-angles of Tyr18 were changed to rotate the side chain hydroxyl group towards ATPγS. We analyzed the complex interface with the PISA server70.
Protein expression and crystallization
A baculovirus vector encoding hexahistidine-tagged ACK1 kinase domain39 was used to transfect Sf9 insect cells using the Bac-to-Bac System (Invitrogen). After 3 rounds of amplification, the recombinant baculovirus was used to infect 600 ml of Sf9 cells at 2 ×106 cells/ml. After three days of infection, the cells were harvested and lysed by two passages through a French pressure cell. The lysis buffer (40 ml) contained 20 mM Tris (pH 8.0), 5 mM beta-mercaptoethanol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1 mM Na3VO4. The lysate was centrifuged at 40,000 × g for 30 min, then filtered successively over 5.0 and 0.8 µm filters. The filtrate was applied to a 3 ml nickel-nitriloacetic acid column using a peristaltic pump. The column was washed first with 150 ml of buffer containing 20 mM Tris (pH 8.0), 2 mM imidazole, 0.5 M NaCl, 10% glycerol, 5 mM beta-mercaptoethanol, and 1 mM Na3VO4. The second wash was with 50 ml of 20 mM Tris (pH 8.0), 1 M NaCl. After a final wash with 20 mM Tris (pH 8.0), Ack1 was eluted with 20 mM Tris buffer containing 100 mM imidazole, 5 mM beta-mercaptoethanol, and 10% glycerol. Fractions containing Ack1 were stored at −80 °C. Kinase activity was confirmed by the phosphocellulose paper binding assay using [32P]-ATP and a peptide based on the phosphorylation site of Wiskott-Aldrich Syndrome Protein (WASP).
The N-terminal His-tag was removed from ACK1 by cleavage with TEV protease. ACK1 was dialyzed at 4 °C against 25 mM HEPES (pH 7.5), 20 mM imidazole, 0.5 mM TCEP, 300 mM NaCl, 10% glycerol, in the presence of His-tagged TEV (20:0.5 ratio). In order to separate TEV protease and the cleaved His-tag peptide, the solution was applied to a second Ni-NTA column. Cleaved ACK1 was eluted using column buffer containing 100 mM imidazole, while the His-tag and TEV remained bound to the resin. The eluate was collected, concentrated and purified further on a Superdex 200 (16/60) Column. The Superdex 200 column was equilibrated using 25 mM Hepes (pH 7.7), 300 mM NaCl, 0.5 mM TCEP, 20 mM MgCl2, and 10% Glycerol. Peak fractions were collected and analyzed by SDS–PAGE. Purified ACK1 was concentrated to 4 mg/ml and stored in size exclusion chromatography buffer at −80 °C.
For the crystallization, 4 mg/mL of purified ACK1 in SEC buffer was used. Purified ACK1 kinase domain was crystallized using the hanging-drop vapor diffusion method at room temperature (18 °C). Protein was incubated with inhibitor (R)-9b stock solution in 1:2 protein – inhibitor molar ratio on ice for ½ h. Incubated complex mixture was spun at 12000 rpm for a short span of 10 min to separate precipitates and aqueous complex solution, before the crystallization drop set up. The best crystals for the complex were obtained in drops that were equilibrated with the reservoir solution consisting of 50 mM Bis-Tris (pH 6.5), 23% (w/v) polyethylene glycol 3350, 100 mM MgCl2, and 2.5% Glycerol in approximately 2 days. Crystals were flash frozen by direct immersion in liquid nitrogen using 20% (v/v) glycerol as the cryoprotectant in reservoir solution.
X-ray diffraction data were collected at NSLS-2 AMX and processed using XDS71 accounting for anisotropic diffraction. The structure was solved by molecular replacement using the structure of ACK1 kinase domain (PDN-entry 4HZR)33 as a search model in Phenix72 using Phaser73. The model was built in COOTt36 and refined in Phenix.
Mice tumor studies
All animal experiments were performed using the standards for humane care in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The WT and Ack1 KO mice used for the experiments were in-bred as described above. The C57BL/6 mice (Strain code: 027, Stock Number: C57BL/6-027) used for the ACK1 inhibitor studies was purchased from Charles River Laboratories, USA. All mice were co-housed with 3–5 mice per cage and maintained in a controlled pathogen-free/germ-free environment with a temperature of 20–23 °C, 12/12 h light/dark cycle, 50–60% humidity, and food and water provided ad libitum. For all mice experiments, tumor volumes of 1500–1700 mm3 or end of treatment period, was considered as the humane end point. The maximum allowed weight loss was 10% of the body weight.
1.2 × 106 TRAMP-C2 cells were suspended in 200 µl of PBS with 50% matrigel (BD Biosciences) and were implanted subcutaneously into the dorsal flank of 6- to 8-weeks old WT and Ack1 KO mice. Tumor volumes were measured twice a week using calipers. Formation of tumors was monitored over an entire 9–11 week period. At the end of the study, when the tumor volumes were approximately 1500–1700 mm3, all mice were humanely euthanized by carbon dioxide inhalation, followed by cervical dislocation. Tumors were extracted and weighed. Additionally, spleen, thymus and femurs were collected and lymph nodes were drained for immunoblotting. Splenocytes and thymocytes were isolated for flow cytometry and tumors were used for further quantitative studies.
To study the antitumor efficacy of (R)-9b, TRAMP-C2 cells were implanted in 6- to 8-weeks old male C57BL/6 mice. 4 weeks postinjection of cells when tumors were palpable, mice were injected subcutaneously with (R)-9b (or vehicle) at the concentrations 24 mg/kg of body weight, five times a week, for 4 weeks. Tumor volumes were measured twice weekly using calipers. At the end of the treatment period, all mice were humanely euthanized by carbon dioxide inhalation, followed by cervical dislocation. The tumors were extracted and weighed. Splenocytes were isolated for flow cytometry and tumors were used for further quantitative studies.
Antibody depletion studies
To determine T-cell mediated growth of TRAMP-C2 tumors, T-cell depletion experiment was performed. WT and Ack1 KO male mice (6–8 weeks) were injected intraperitoneally with 250 μg/mouse αCD4 (GK1.5, BioXcell) or 250 μg/mouse αCD8β (53-5.8, BioXcell) or IgG (control). Three days post the antibody injection, mice were subcutaneously implanted with 1.5 × 106 TRAMP-C2 cells that were suspended in 200 µl of PBS with 50% matrigel (BD Biosciences). Following tumor implantation, mice were injected with the above antibodies intraperitoneally, once a week, for 5 weeks. Tumor growth was monitored and measured with calipers. At the end of the treatment period, all mice were humanely euthanized by carbon dioxide inhalation, followed by cervical dislocation. Tumors were extracted and weighed.
Adoptive transfer experiment
To determine tumor specific T cell responses, TRAMP-C2 tumors were implanted subcutaneously in 10–12 weeks old NSG mice purchased from the Jackson Laboratory (Strain number: 005557, JAX stock #005557; IACUC protocol no 20-0383). T cells were purified from the splenocytes of WT and Ack1 KO mice. Purified T cells (1.5 × 106) resuspended in PBS were injected once a week from the 4th day after tumor implantation. After 6 weeks, mice were humanely euthanized by carbon dioxide inhalation followed by cervical dislocation. Splenocytes were isolated and lymph nodes were drained. The T cell phenotype and levels of SPAS-1 expression were assessed. Splenocytes or cells from drained lymph nodes were stained with Live/Dead Aqua (1:800), anti-CD3 PECy7 (1:400), anti-CD8 APC (1:400), anti-CD4 Pacific blue (1:400), anti-CD44 PE (1:400) and anti-CD62L PerCP-Cy5.5 (1:400) or SPAS-1 PE (1:550) or control tetramer followed by flow cytometry. Tumors were excised. For the analysis of TILs, tumors were dissociated in digestion media and a single cell suspension was made. TILs were stained with the above-mentioned antibodies along with anti-CD45 APC Cy7 (1:300) followed by flow cytometry. The persistence of the transferred T cells was assessed by injecting CFSE-labeled T cells in the NSG mice and T-cell proliferation was assessed periodically by isolating lymphocytes from blood collected by sub-mandibular puncture, followed by flow cytometry up to 14 days post adoptive transfer.
ICB combination studies
To evaluate the effect of (R)-9b as a combination drug with ICB, 1.5 × 106 TRAMP-C2 cells were suspended in 200 µl of PBS with 50% matrigel (BD Biosciences) and were implanted subcutaneously into the dorsal flank of 6- to 8-weeks old male C57BL/6 mice. Mice were injected intraperitoneally with combination of anti–CTLA-4 [clone 9H10; BioXCell] and anti–PD-1 [RMP1–14; BioXCell] (100 μg each) antibodies (ICB) on days 32, 34, and 41. In the second set, once the tumors reached approximately 125 mm3 in size, mice were injected with (R)-9b in 6% captisol in PBS at the concentration of 24 mg/kg of body weight, five times a week, for 4 weeks. The third set of mice received ICB antibodies in combination with subcutaneous injections of (R)-9b five times a week, for 4 weeks. Tumor volumes were measured twice a week using calipers. At the end of the treatment period, all mice were humanely euthanized by carbon dioxide inhalation, followed by cervical dislocation. Tumors were extracted and weighed. Spleen lysates were used for immunoblotting. Splenocytes were isolated and stained with Live/Dead Aqua (1:800), anti-CD4 Pacific blue (1:400), anti-CD25 FITC (1:250). Cells were then permeabilized and intracellular staining was done with anti-FoxP3 PE (1:250) followed by flow cytometry.
Collection of human prostate tissue samples for organoid studies
Human prostate tumor and adjacent normal tissue samples were obtained following radical prostatectomy with patient’s consent under the IRB-approved GU Banking Protocol (HRPO #201411135). Normal (i.e., far from the tumor site) and tumor tissue (i.e., the center core of cancerous lesion) were identified by magnetic resonance imaging (MRI) studies and pathology reports. Tissues were dissected as per the above identification by a pathologist within 30 min of surgery to avoid ischemia. Prostate tissue specimens were collected immediately on ice, and a 1 mm core was fixed in formalin, embedded to be reviewed again by the board-certified pathologist (CW), who assigns a Gleason score to all specimen collected using this procedure. The period of tissue storage was no longer than 1 hr from surgery to culture to maintain organoid viability. Prostate tissues were washed twice with cold 1X Phosphate buffered saline and dissected into equal 3–5 mm pieces for molecular profiling and organoid culture as described below.
Generation of human prostate derived tumor organoids (PDTO) from fresh tissues
To generate normal and tumor organoids, fresh tissues of normal/tumor origin from radical prostatectomies were washed twice with 1X PBS and minced into approximately 0.1–0.5 mm diameter pieces with disposable sterile surgical scalpel blades. The minced tissues were transferred to 2 ml of collagenase containing cell dissociation media and incubated at 37 °C for 45 min with continuous gentle rotation. The Cell dissociation media was prepared as follows: A 5 mg of Collagenase (Collagenase Type II, Gibco, no. 17101-015) was added to 1 ml of basal advDMEM/F12 (Advanced DMEM/F12, Invitrogen, no. 12634-034) containing penicillin/streptomycin (Gibco, no. 10378016), 10 mM HEPES (Invitrogen, no. 15630-056) and 2 mM GlutaMAX (Invitrogen, no. 35050-079) and 10 μM of ROCK inhibitor Y27632 (Sigma, no. Y0503) and filter sterilized and stored for 3 days at 4 °C74.
The dissociated prostate tissues were centrifuged at 500 g for 5 min at 4 °C, and the pellet was washed with ice-cold basal advDMEM/F12 media. The pelleted dissociated tissues were trypsinized in 5 ml of TrypLE (Gibco, no. 12605-028) with Y-27632 for 20 min with intermittent pipetting every 10 min. Cell suspension was washed with 10 ml of ice-cold basal advDMEM/F12 media. The dissociated cell suspension was filtered with a 40-μm mesh filter. Cells were counted and 1 × 104 cells were resuspended in 75% Matrigel (Corning) and plated in a 40 μl drop in the middle of one well of a 24 well plate. The plate was turned upside down and incubated for 30 min at 37 °C and 5% CO2. A 0.5 ml of complete human prostate organoids culture media containing B27-serum free (Life technologies), Nicotinamide and N-acetylcysteine (Sigma Aldrich), Noggin (PeproTech), R-Spondin (R&D systems-Cultrex®), p38 MAP kinase inhibitor SB202190 (Sigma-Aldrich), epidermal growth factor (EGF), fibroblast growth factor10, FGF-10 and FGF2 (PeproTech), TGFβ kinase/activin receptor-like kinase (ALK5) inhibitor A83-01(Sigma-Aldrich), prostaglandin E2 (Tocris Bioscience) and Y-27632 (Abmol Biosciences) culture media was added after the matrigel solidification. Cells were maintained under this condition till the PDTOs achieved 300-micron size. The media was replenished every 3–4 days to maintain the growth integrity of PDTOs and passaged regularly after 3–4 weeks.
Organoids were characterized; briefly, the tissues (the starting material) and the organoids were reviewed by a board-certified pathologist (histological examination post-H&E staining). In addition to histology, we performed quantitative RT-PCR analysis for well-known markers of prostate cancer, e.g. androgen receptor (AR), HOXB13, and PSA/KLK3. The tumor organoids showed a significant upregulation of these markers compared to normal, validating the pathologist’s analysis.
C4-2B cells were seeded at approximately 5000 cells per well (24-well plate) into 40 μl growth factor reduced and phenol red-free Matrigel (Corning). C4-2B spheroids were maintained in organoid culture media and used for further experiments.
Co-culture of (R)−9b treated PBMCs with human 3D prostate model systems
To assess the cell killing effect of (R)-9b treated peripheral blood mononuclear cells (PBMCs) on human 3D prostate organoid model systems, PBMCs were treated with (R)-9b (1 µM) and the compound was washed. Post 24 h co-culturing with (R)-9b treated PBMCs, the PDTOs or C4-2B spheroids were treated with dispase II (STEMCELL Technologies), followed by incubation for 60 min at 37 °C to digest the Matrigel. PDTOs and C4-2B spheroids were washed twice with ice-cold basal advDMEM/F12 media. To the pellet, 10 µl of binding buffer with propidium iodide and Hoechst was added and incubated for 10 min at room temperature in the dark. The entire content was transferred to a microscopic slide, and fluorescent images were taken in EVOS M5000 imaging system (Thermofisher Scientific).
Recombinant DNA transfection
HEK293T cells were transfected with control pcDNA3.1 (Vector) ACK1, cACK1, ACK-KD, LCK, CSK or CSK-Y18F using X-tremeGENE HP DNA transfection reagent (Sigma). Transfected cells were grown in DMEM with 10% fetal bovine serum for 48 hr and/or treated with inhibitors/drugs and harvested for immunoblotting as per experimental conditions.
Generation and affinity purification of the pY18-CSK antibody
Two CSK peptides coupled to immunogenic carrier proteins were synthesized as shown below, and pTyr18-CSK antibodies were custom synthesized by GenScript, NJ.
CSK Phosphorylated peptide: IAKpYNFHGTAEQDL
CSK peptide: IAKYNFHGTAEQDL
Two rabbits were immunized twice with the phosphopeptide, and the sera from these rabbits was affinity purified. Two antigen-affinity columns were used to purify the phospho-specific antibodies. The first column was the nonphosphopeptide affinity column. Antibodies recognizing the unphosphorylated residues of the peptide bound to the column. The flow-through fraction was collected and then applied to the second column, the phosphopeptide column. Antibodies recognizing the phospho-Tyr residue bound to the column and were eluted as phospho-specific antibodies.
Knockdown of ACK1 by siRNA Interference
Jurkat cells were transfected with Ack1-specific siRNA (Dharmacon RNA Technologies, Lafayette, CO) or control siRNA by using X-tremeGENE transfection reagent (Sigma-Aldrich). Cells were allowed to culture for 48 hr and then harvested for immunoblotting experiments.
Spleen and thymus were collected from the WT and Ack1 KO mice. Organs were homogenized and lysed by sonication in receptor lysis buffer (RLB) containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 1% Triton X-100, 1 mM DTT, 10% glycerol, phosphatase inhibitors (50 mM NaF, 1 mM Na2VO4), and protease inhibitor mix (Roche). PBMCs from prostate cancer patients, TRAMP-C2, C4-2B, HEK293T and Jurkat cells were transfected and/or treated as per experimental requirement and were harvested and lysed by sonication in RLB. Lysates were quantitated and 20 to 100 µg of protein lysates were boiled in SDS sample buffer, size fractionated by SDS-PAGE, and transferred onto a PVDF membrane (GE Healthcare). After blocking in 3% bovine serum albumin (BSA), membranes were incubated with the following primary antibodies: anti-ACK1 (1:1000), anti-H3 (1:1000), Anti-PD-1(1:1000), anti-CSK (1:1000), anti-FLAG (1:1000), anti- phosphoTyrosine (pTyr) (1:500), anti-LCK (1:1000), anti-Ub (1:1000), anti-LCK pY505 (1:1000), anti-LCK pY394 (1:1000), anti-LAT pY132 (1:1000), anti-ZAP-70 pY319 (1:1000), anti-PLC-g pY783 (1:1000), anti-ACK1 pY284 (1:1000), anti-PAG1 (1:1000), anti-HA (1:1000), anti-EZH2 (1:1000), anti-Actin (1:9,000). Following three washes in PBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibody. The blots were washed thrice and the signals visualized by enhanced chemiluminescence (ECL) system according to manufacturer’s instructions (Thermo Scientific) using Invitrogen™ iBright™ FL1000 imaging system.
For immunoprecipitation studies, cell or organ tissue were lysed by sonication in RLB, the lysates were quantitated and 0.5 to 1 mg of protein lysate was immunoprecipitated using 2 μg of anti-CSK, anti-pTyr, anti-FLAG, anti-HA, anti-Myc or anti-EZH2 antibody coupled with protein A/G sepharose (Santacruz) overnight, followed by washes with RLB and PBS buffers. The beads were boiled in sample buffer and immunoblotting was performed as described above. Densitometric analysis using ImageJ software (ImageJ, NIH, USA) was performed for each representative immunoblot image and actin normalized relative fold change intensity for each lane is incorporated, wherever required.
HEK293T (2.2 × 106) cells were co-transfected with ACK1 or vector. Post 48 hr of transfection, cells were processed for LC-MS/MS analysis. Samples were digested overnight with modified sequencing grade trypsin (Promega, Madison, WI), Glu-C (Worthington, Lakewood, NJ), or Arg-C (Roche,Switzerland). Phosphopeptides were enriched using Phospho Select IMAC resins (Sigma). A nanoflow ultra high performance liquid chromatograph (RSLC, Dionex, Sunnyvale, CA) coupled to an electrospray bench top orbitrap mass spectrometer (Q-Exactive plus, Thermo, San Jose, CA) was used for tandem mass spectrometry peptide sequencing experiments. The sample was first loaded onto a pre-column (2 cm × 100 μm ID packed with C18 reversedphase resin, 5 μm, 100 Å) and washed for 8 min with aqueous 2% acetonitrile and 0.04% trifluoroacetic acid. The trapped peptides were eluted onto the analytical column, (C18, 75 μm ID × 50 cm, 2 μm, 100 Å, Dionex, Sunnyvale, CA). The 90-minute gradient was programmed as: 95% solvent A (2% acetonitrile + 0.1% formic acid) for 8 min, solvent B (90% acetonitrile + 0.1% formic acid) from 5% to 38.5% in 60 min, then solvent B from 50 to 90% B in 7 min and held at 90% for 5 min, followed by solvent B from 90 to 5% in 1 min and re-equilibrate for 10 min. The flow rate on analytical column was 300 nl/min. Sixteen tandem mass spectra were collected in a data-dependent manner following each survey scan. Both MS and MS/MS scans were performed in Orbitrap to obtain accurate mass measurement using 60 second exclusion for previously sampled peptide peaks. Sequences were assigned using Sequest (Thermo) and Mascot (www.matrixscience.com) database searches against SwissProt protein entries of the appropriate species. Oxidized methionine, carbamidomethyl cysteine, and phosphorylated serine, threonine and tyrosine were selected as variable modifications, and as many as 3 missed cleavages were allowed. The precursor mass tolerance was 20 ppm and MS/MS mass tolerance was 0.05 Da. Assignments were manually verified by inspection of the tandem mass spectra and coalesced into Scaffold reports (www.proteomesoftware.com).
Under sterile conditions, spleen, lymph nodes and femurs were harvested from naïve or TRAMP-C2 tumor bearing WT and Ack1 KO mice. Single cells were made and RBCs were lysed using ACK lysis buffer. For immunophenotyping, 1 × 106 cells were incubated with Live/Dead Aqua (1:800), anti-CD3 PECy7 (1:400), anti-CD4 Pacific blue (1:400), anti-CD8 APC (1:400) antibodies to identify T-cells. The anti-CD19 PerCP-Cy 5.5 antibody (1:400) was used to identify B-cells, anti-NK1.1 PE antibody (1:500) was used to identify NK cells, anti-CD4 Pacific blue (1:400), anti-CD25 FITC (1:250) and intracellular anti-FoxP3 PE (1:250) antibodies were used to identify regulatory T cells, anti-CD11b PerCP-Cy 5.5 (1:400) and anti-Gr-1 APC (1:400) antibodies were used to identify subset of myeloid cells. Antibodies were incubated for 20 min, according to manufacturer’s instructions (BD biosciences and BioLegend). T-cells were purified from splenocytes using mouse CD3+ T Cell Enrichment Column (R&D Systems) according to manufacturer’s protocol. Purified T-cells or splenocytes were stained with Live/Dead Aqua (1:800) and either anti-CD3 PECy7 (1:400), anti-CD8 APC (1:400) and activation markers – anti-CD137 FITC (1:400), anti-CD44 PE (1:400) and anti-CD62L PerCP-Cy5.5 (1:400) or exhaustion markers – anti-PD1 FITC (1:400), anti-Lag3 PerCP-Cy5.5 (1:400), anti-Tim3 PE (1:400) or anti-CD4 Pacific blue (1:400) with activation marker anti-CD69 PE (1:400) antibodies. Cells were then permeabilized and intracellular staining was done with anti-perforin FITC (1:300), anti-IL2 APC-Cy7 (1:300) or anti-IFN gamma BV786 (1:300) antibodies. Tumor infiltrating T cells were identified by staining with Live/Dead Aqua (1:800), anti-CD45 APC-Cy7 (1:300), anti-CD3 PECy7 (1:400), anti-CD8 APC (1:400), anti-CD4 Pacific blue (1:400), anti-CD69 PE (1:400), anti-CD25 FITC (1:250) antibodies. Cells were then permeabilized and intracellular staining was done with anti-perforin FITC (1:300) and anti-FoxP3 PE (1:250) antibodies. Parallel immunophenotyping of spleen and tumor draining lymph nodes was performed in control and (R)-9b treated tumor bearing C57BL/6 mice staining with Live/Dead Aqua (1:800), anti-CD3, anti-CD4, anti-CD8, activation markers, exhaustion markers and intracellular staining with antibodies for markers as mentioned above. Samples were analyzed using BD FACSCanto II or LSR Fortessa (BD Biosciences) and post-acquisition analysis was done using FlowJo software (Tree Star Inc).
Inhibition of pY18-CSK upon ACK1 knockdown or pharmacological inhibition was assessed by flow cytometry. Briefly, Jurkat cells treated with vehicle or (R)-9b and splenocytes from WT and Ack1 KO mice were incubated with pTyr18-CSK primary antibody. Cells were washed, incubated with anti-rabbit Alexa Fluor® 488 antibody, fixed and flow cytometry was performed.
Human PBMC separation
Whole blood was collected from healthy volunteers and prostate cancer patients in VACUTTE® tubes coated with sodium heparin. PBMC was isolated using Lymphocyte Separation Medium from Corning according to manufacturer’s protocol.
Intracellular calcium measurements
Jurkat cells were incubated with 1 μM (R)-9b for 3 h and 6 h and washed with PBS. Similarly, splenocytes isolated from C57BL/6 mice were treated with 1 μM (R)-9b for 6 h. Cells were loaded with Fluo-8 (Abcam) and incubated for 30 min at 37 °C. Intracellular calcium flux was measured using flow cytometry. Ionomycin was added at 300th second. Splenocytes and thymocytes isolated from WT and Ack1 KO mice were incubated with TRAMP-C2 cells for 6 h and washed with PBS. Splenocytes from C57BL/6 mice were isolated and treated with (R)-9b for 6 h, washed and incubated with TRAMP-C2 cells for 6 h. Intracellular calcium flux measurement was done as described above. PBMCs isolated from healthy donors, CRPC and mHSPC patients were incubated with 1 μM (R)-9b for 6 h, washed and calcium flux was measured as described above. For the analyzing the response of (R)-9b treated cells upon anti-CD3 addition, dye-loaded cells were recorded for initial 30 s and then monitored after addition of anti-CD3 antibodies, followed by ionomycin addition.
All RT reactions were done at the same time so that the same reactions could be used for all gene studies. For the construction of standard curves, serial dilutions of pooled sample RNA were used (50, 10, 2, 0.4, 0.08, and 0.016 ng) per reverse transcriptase reaction. One “no RNA” control and one “no Reverse Transcriptase” control was included for the standard curve. Three reactions were performed for each sample: 10 ng and a NoRT (10 ng) control. Real-time quantitative PCR analyses were performed using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). All standards, the no template control (H2O), the No RNA control, the no Reverse Transcriptase control were tested in six wells per gene (2 wells/plate x 3 plates/gene). All samples were tested in triplicate wells each for the 10 ng concentrations. The no RT controls were tested in duplicate wells. PCR was carried out with SYBR Green PCR Master Mix (Applied Biosystems) using 2 µl of cDNA and the primers in a 20-µl final reaction mixture. After 2-min incubation at 50 °C, AmpliTaq Gold was activated by 10-min incubation at 95 °C, followed by 40 PCR cycles consisting of 15 s of denaturation at 95 °C and hybridization of primers for 1 min at 55 °C. Dissociation curves were generated for each plate to verify the integrity of the primers. Data were analyzed using SDS software version 2.2.2 and exported into an Excel spreadsheet. The actin or 18s rRNA data were used for normalizing the gene values; i.e., ng gene/ng actin or 18s rRNA per well. The primer sequences are shown in Supplementary Table 2.
Formalin-fixed paraffin-embedded (FFPE) tissue blocks were made from TRAMP-C2 tumors treated with Veh+IgG, ICB, (R)-9b and ICB + (R)-9b. Immunostaining was done on sections (4 μm) of the tissue blocks, which was mounted on glass slides and dried in an oven at 60 °C for 1 h. The antigen retrieval was done for 20 mins with Tris based antigen retrieval solution (pH 9). Slides were incubated with Rabbit anti-mouse CD3 (E4T1B) antibody (Cell Signaling Technologies, USA). The mounted slides were analyzed under the bright field microscope and the total number of CD3 cells per field was calculated.
C4-2B cells (5 × 107 cells) were either treated with vehicle or (R)-9b. Total RNA was extracted using RNeasy kit from QIAGEN (Cat #74136). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 TapeStation. Library preparation was performed with 5 to 10ug of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 min. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer’s instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3’ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12–15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Base calls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read. RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1. Gene counts were derived from the number of uniquely aligned unambiguous reads.
Chromatin immunoprecipitation (ChIP)
C4-2B and TRAMP-C2 cells (5 × 107 cells) were either treated with vehicle or (R)−9b. Cells were harvested, fixed in 1% formaldehyde. Cell pellets were resuspended in RLB buffer and sonicated. The soluble chromatin was incubated at 4 °C with antibodies and protein-G and -A magnetic beads. The soluble chromatin was processed in the same way without immunoprecipitation and termed input DNA. The complexes were washed with RLB buffer, followed by ChIP buffer 1 and 2 (Active Motif), eluted with elution buffer and subjected to proteinase-K treatment. Fixed ChIP DNA was ‘reverse’ cross linked and subjected to proteinase-K treatment and purified using PCR DNA purification columns (Qiagen). The purified ChIP DNA was validated by real-time PCR as described above.
Levels of IFN-γ in the blood serum of WT and Ack1 KO mice and cell culture supernatant of splenocytes from C57BL/6 mice treated overnight with (R)-9b was measured using mouse Interferon-γ ELISA Kit (R&D Systems) according to manufacturer’s protocol.
Cell mediated cytotoxicity assay
Splenocytes isolated from WT and Ack1 KO mice were incubated with TRAMP-C2 cells. After 24 h, cells were labeled with 7-AAD (Biolegend) and the percentage of 7-AAD+ cells were evaluated by flow cytometry. In addition, splenocytes were isolated from C57BL/6 mice, treated overnight with 1 μM (R)-9b. Cells were washed once with PBS and incubated with TRAMP-C2 cells, pre-stained with CFSE (BioLegend). After 24 h, cells were labeled with 7-AAD (Biolegend) and cell lysis was evaluated using flow cytometry analyzing the percentage of CFSE+ 7-AAD+ cells.
All data are presented as mean ± SEM and all statistical parameters and analysis are mentioned in the figure legends respectively. Data for all experiments were analyzed with GraphPad Prism 8.0 software. All statistical analyses were performed using Student t-test unless otherwise specified. p values less than 0.05 were considered as statistically significant.
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