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Clinical Research in HIV or AIDS

Dasatinib: Effects on the Macrophage Phospho Proteome with a Focus on SAMHD1 and HIV-1 Infection

Research Article | Open Access

  • 1. Department of Pathology, University of Utah School of Medicine, USA
  • 2. AIDS Immunopathology Unit, National Center of Microbiology, Spain
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Corresponding Authors
Vicente Planelles, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA
Abstract

Macrophages are one of the main cellular targets of human immunodeficiency virus type 1 (HIV-1). Macrophage infection by HIV-1 is inefficient due to the presence of the viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1). Ex vivo, human monocyte- erived macrophages (MDMs) express SAMHD1 in an equilibrium between active (un-phosphorylated) and inactive (phosphorylated) states. Others and we have shown that treatment of MDMs with the FDA approved tyrosine kinase inhibitor, dasatinib, ablates SAMHD1 phosphorylation, thus skewing the balance towards a cellular state that is refractory to HIV-1 infection. We hypothesized that dasatinib inhibits a putative tyrosine kinase that is upstream of SAMHD1. In the search for this tyrosine kinase, we probed several candidates and were unable to identify a single target that, when inhibited, was sufficient to explain the dephosphorylating of SAMHD1 we observe upon treatment with dasatinib. On the other hand, we probed the ability of dasatinib to directly inhibit the serine/threonine cyclin dependent kinases 1, 2, 4 and 6 and confirmed that dasatinib directly inhibits these kinases. Therefore, our results show that inhibition of the proximal CDKs 1, 2, 4 and 6 by dasatinib is clearly detectable, leads to blockade of infection by HIV-1, and may be sufficient to explain the activity of dasatinib against SAMHD1 phosphorylation.

Keywords

SAMHD1, HIV-1, Phosphoproteome, CDK1, CDK4, Macrophages, Cell cycle, Dasatinib, Tyrosine kinase, Serine/threonine kinase

Cite this article

Williams ESCP, Szaniawski MA, Martins LJ, Innis EA, Alcamí J, et al. (2022) Dasatinib: Effects on the Macrophage Phospho Proteome with a Focus on SAMHD1 and HIV-1 Infection. Clin Res HIV/AIDS 8(1): 1053.

INTRODUCTION

Macrophages and other myeloid cells are long lived and play well-established roles during initial virus infection, viral persistence during antiretroviral treatment, as well as contributing to the spread of HIV-1 to the central nervous system (CNS) [1-3]. Infection of macrophages by HIV-1 is inefficient due to the presence of viral restriction factor sterile alpha motif and histidine aspartic acid domain containing protein 1 (SAMHD1) [4,5]. SAMHD1 is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase that regulates the concentration of dNTPs within cells [6], and when active, maintains dNTP levels below the threshold necessary to support efficient reverse transcription [6-10]. Regulation of SAMHD1 phosphorylation is cell cycle dependent and, in post-mitotic macrophages, SAMHD1 is primarily found in an active (unphosphorylated) state, rendering myeloid lineage cells resistant to HIV-1 infection [11-16]. SAMHD1 phosphorylation/dephosphorylation levels are also responsible for the lower permissivity of female-derived human macrophages compared to their male-derived counterparts [17].

Although many cyclin-dependent kinases (CDK) have been shown to contribute to the phospho-regulation of SAMHD1 [15,18-22], CDKs are serine/threonine kinases whereas dasatinib was identified as a tyrosine kinase inhibitor. Therefore, it is tempting to speculate that a putative upstream tyrosine kinase, which is presumably targeted by dasatinib, ultimately controls the activities of downstream CDKs acting on SAMHD1.

In addition to being cell cycle dependent, SAMHD1 phosphorylation can be modulated in vitro by treatment with γC cytokines [23], interferons (IFNs) [21], and multiple tyrosine kinase inhibitors (TKI) [21,24,25]. One TKI, dasatinib, decreases SAMHD1 phosphorylation levels in macrophages to a much greater degree than others tested [21,24].

Dasatinib is a multi-kinase inhibitor that was designed to target Abl and Src family kinases and is used clinically to treat chronic myelogenous leukemia and acute myeloid leukemia [26- 29]. More recently, it has been shown that dasatinib inhibits HIV1 infection in CD4+ T cells [24], and in macrophages [21], due to its ability to reduce SAMHD1 phosphorylation. However, the specific mechanism through which dasatinib decreases SAMHD1 phosphorylation levels is unknown. Our previous studies demonstrated that the mechanism through which interferons act to induce dephosphorylation of SAMHD1 is different from the pathway inhibited by dasatinib [21]. While types I and II IFN induce dephosphorylation of SAMHD1 in part by diminishing transcription of the CDK1 gene, this change was not observed in dasatinib treated cells [21].

We hypothesized that a putative upstream tyrosine kinase may be a master regulator of several serine/threonine kinases, which collectively can phosphorylate residue threonine-592 of SAMHD1. To begin steps toward identifying a putative upstream tyrosine kinase, we resorted to a previous study [30], that identified multiple protein tyrosine kinases and serine/threonine kinases as being inhibited by dasatinib in addition to Abl and Src. We used specific kinase inhibitors to probe the potential roles of several upstream kinases.

MATERIALS AND METHODS

Isolation of healthy donor PBMC

Healthy volunteer donors aged 18 and above were recruited for this study to undergo peripheral phlebotomy in accordance with the University of Utah institutional review board (IRB) protocol 00067637. Peripheral blood mononuclear cells (PBMCs) were isolated from total blood via Lymphoprep density gradient as previously described [21].

Generation and infection of MDM

Following collection of PBMCs, CD14+ monocytes were isolated through positive selection using magnetic beads (Miltenyi Biotec, US). Cells were plated in untreated 24-well plates at a density of 6x105 in serum free Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2mM L-glutamine (Gibco, US), and 1% Pen-Strep (Gibco, US) and allowed to adhere for greater than 2 hours. Serum-free medium was then gently removed and replaced with 600μL RPMI 1640 medium supplemented as above, with the addition of 5% pooled human serum (Innovative Research) and 5% fetal bovine serum (FBS) (Gibco, US) (culture medium). The culture medium was changed again on day 5 and experiments began on day 6. MDMs were infected on day 7 with 500ng of p24 of replication competent either HIV-1-NL4-3-BAL-HSA or HIV-1-NL4-3-AD8 viruses diluted in 200-300μL culture medium for 6 hours. Following 6 hours of infection, the total medium volume was brought back up to the culturing volume of 600μL. On day 8, the 600μL of medium containing virus was removed, the cells washed with 1mL of PBS to remove any cell-free virus and replaced with 600μL of fresh culture medium.

Generation and quantification of viruses

Replication competent viruses were generated using calcium phosphate-mediated transfection of HEK293FT cells. Briefly, HEK293FT cells were plated in T175 tissue culture coated flasks (VWR) in 20mL of Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, US) supplemented with 10% FBS (Gibco, US), and 2mM L-glutamine (Gibco, US). The following morning, the medium was replaced, and cells were transfected with 25ug of viral plasmid for 6 hours. The HIV-1-NL4-3-BAL-HSA (HIV-1 BAL) virus was generated using the pNL-43-BAL-IRES-HSA plasmid, and the HIV-1-NL4-3-AD8 (HIV-1 AD8) virus was generated using the pNL4-3-AD8 plasmid. After 6 hours, the transfection medium was replaced, and cells were cultured for 2 days. At which time the virus-containing supernatants were removed, filtered at 0.45μm to remove any cellular debris, and stored at - 80°C until further use. All viruses were quantified by p24 ELISA (Zeptometrix).

Inhibitors

Inhibitors were purchased from suppliers (listed in supplementary table X) and re-suspended at either 10mM or 100mM depending on accompanying documentation. MDMs were initially treated with inhibitors on day 6 post isolation. On day 7, MDMs were infected with 500ng of either HIV-1 BAL or HIV-1 AD8, in the presence of inhibitors. On day 8, the infection medium was removed and replaced with fresh culture medium containing the inhibitors. Inhibitors were tested in MDMs from at least two donors, at multiple concentrations. Determination of effect on SAMHD1 phosphorylation was visualized by western blot and flow cytometry.

Western blotting

MDMs were lysed for western blotting 8 days post isolation. Culture medium was removed, cells were washed with 1mL of ice-cold PBS, following wash, 125μL of ice-cold NETN lysis buffer containing protease and phosphatase inhibitors (complete ULTRA tablets and Postop, by Roche, respectively). Cells lysed on ice for 10 minutes before protein was collected and transferred to Eppendorf tubes. Lysate remained on ice for a minimum of 1 hour, after which they were sonicated 4 times for 15 seconds, to prevent the water in the solicitor from heating. Following sonication, the lysates were centrifuged at 13000 rpm at 4°C for 10 minutes. After sonication, the lysates were transferred to new Eppendorf tubes, and frozen at -20 °C until needed. The protein concentration of each lysate was determined by BCA assay (Pierce BCA Protein Assay Kit, Thermo Scientific) according to manufacturer’s protocol. 10μg of protein was loaded into each well of each gel (Criterion TGX Precast Gels 4-15%, with either 18 or 26 wells, Bio-Rad). Gels were run between 60 and 100 volts and transferred to 0.45 μm PVDF transfer membrane (Thermo Fisher) through semi dry transfer method at 20 volts and 0.4 amps for 30 minutes.

Flow cytometric analysis

MDMs were stained in their 24-well plate to maintain SAMHD1 phosphorylation. Culture medium was removed, and cells were washed with 1mL of PBS. Cells were stained in 200μL PBS at 4°C for 30 minutes with a fixable viability dye (Thermo Fisher) in the presence of an Fc receptor blocking reagent (Miltenyi Biotec) and with or without an anti- mouse heat stable antigen (HSA, CD24) stain (Thermo Fisher) depending on which virus as used to infect cells during each experiment. After 30 minutes, 1mL of PBS was added to each well to dilute the stains and wash the cells. The PBS was removed, and cells were fixed in 200μL of prewarmed BD Cytofix fixation buffer (Becton Dickinson) at 37°C for 15 minutes. Cells were then washed as described in the previous step. Then the PBS was removed, and cells were permeabilized in 200μL of room temperature BD Phosflow perm buffer III (Becton Dickinson) at 4°C for 30 minutes. Cells were then washed with 1mL 1X BD Perm/Wash buffer (Becton Dickinson) as described. After the perm/wash was removed, cells were stained at 4°C for 60 minutes in 200μL perm/wash for phospho-SAMHD1-T592 (Cell Signaling Technologies) with or without a stain for the HIV-1 core antigen, clone KC57 (Beckman Coulter) depending on which virus was used during each experiment. After 1 hour, 1mL of PBS was added to each well and plates were left at 4 °C overnight. The following morning, cells were scraped from the well surface with mini cell scrapers and transferred to flow tubes (Corning). 2mLs of PBS was added to each tube and cells were centrifuged at 5000 rpm for 5 minutes. Following centrifugation, the majority of PBS was aspirated out of each tube, leaving cells pelleted in approximately 250μL. Samples were run using an Attune NxT Cytometer (Thermo Fisher) and analyzed using FlowJo 10.7.1 (Becton Dickinson).

RESULTS AND DISCUSSION

Dasatinib was originally designed to inhibit the tyrosine kinases Abl and Src to treat imatinib- resistant chronic myelogenous leukemia and Philadelphia chromosome-positive acute myelogenous leukemia [26]. Therefore, we first tested whether inhibition of either Abl or Src would result in lower phosphorylation levels of SAMHD1 in macrophages. We confirmed that dasatinib inhibits both Abl and Src (Figure 1A). Next, we inhibited Abl and Src individually to see if their inhibition caused a reduction in SAMHD1 phosphorylation. Figure 1B shows that when we treated cells with the Abl-specific inhibitor, flumatinib [31], we did not observe a change in SAMHD1 phosphorylation. Additionally, when we used the Src inhibitor 1 [32], SAMHD1 phosphorylation was also unchanged (Figure 1C).

A: Dasatinib inhibits Abl and Src phosphorylation. B: Flumatinib, Abl specifici inhibitor, does not reduce SAMHD1 phosphorylation. C:  Inhibiting Src, with Src inhibitor I, does not inhibit SAMHD1 phosphorylation.

Figure 1: A: Dasatinib inhibits Abl and Src phosphorylation. B: Flumatinib, Abl specifici inhibitor, does not reduce SAMHD1 phosphorylation. C: Inhibiting Src, with Src inhibitor I, does not inhibit SAMHD1 phosphorylation.

 

After ruling Abl and Src out as upstream players in SAMHD1 phosphorylation, we decided to test a broader panel of tyrosine kinases for their potential roles in mediating downstream SAMHD1 phosphorylation. We tested inhibitors of vascular endothelial growth factor receptor VEGFR), platelet-derived growth factor receptor (PDGFR), ephrin receptor (EphR), epidermal growth factor receptor (EGFR), and fibroblast growth factor receptor (FGFR) (Figure 2).

Inhibition of cell surface tyrosine kinase receptors in macrophages does not reduce SAMHD1 phosphorylation to the same degree as  dasatinib.

Figure 2: Inhibition of cell surface tyrosine kinase receptors in macrophages does not reduce SAMHD1 phosphorylation to the same degree as dasatinib.

 

Inhibiting each of these tyrosine kinases individually did not reduce SAMHD1 phosphorylation to any degree close to that observed when macrophages were treated with dasatinib. We also tested Janus kinases for their potential roles in regulating SAMHD1 phosphorylation.

Inhibiting the JAK proteins does not inhibit SAMHD1 phosphorylation to the same degree as dasatinib.

Figure 3: Inhibiting the JAK proteins does not inhibit SAMHD1 phosphorylation to the same degree as dasatinib

 

Figure 3 shows that dasatinib induces dephosphorylation in JAK1, 2 and 3, and tyrosine kinase 2 (TYK2). However, inhibiting any of these four JAKs individually failed to reduce SAMHD1 phosphorylation. Several reports support CDK1 as the sole kinase responsible for SAMHD1 phosphorylation [18,20,21,24,33], although other reports propose that CDK1 acts in combination with cyclin A2 [18,20,24]. Yet, other reports propose that CDK2 alone [20,34], or in conjunction with CDK6 [15], or cyclin D3 [35], can phosphorylate SAMHD1. Still others suggest that CDK6 works with CDK4 to phosphorylate SAMHD1 [36], or that CDK4 under the control of cyclin D2 is responsible [37]. Our inability to identify upstream tyrosine kinases that could modulate SAMHD1 phosphorylation in response to dasatinib treatment could potentially be explained if dasatinib had a direct inhibitory effect on the cyclin dependent kinases CDK 1, 2, 4 and 6.Therefore, we tested this idea in Figure 4, which shows that dasatinib induces dephosphorylation of CDK1 at residue Threo161, which is synonymous with CDK1 inactivation.

Dasatinib inhibits the phosphorylation of CDK1 at threonine 161, in the kinase loop, of monocyte-derived macrophages.

Figure 4: Dasatinib inhibits the phosphorylation of CDK1 at threonine 161, in the kinase loop, of monocyte-derived macrophages.

 

We then tested whether specific inhibition of CDK1, CDK2, CDK4 and CDK6 with CDK1 inhibitor, CDK1/2 inhibitor III, CDK4 inhibitor, and CDK4/6 inhibitor IV (respectively) would reduce SAMHD1 phosphorylation. Individual inhibition of each of the cyclin dependent kinases 1, 2, 4 and 6 indeed reduced SAMHD1 phosphorylation (Figure 5A). All CDK inhibitors tested uniformly reduced phosphorylation of SAMHD1 (Figure 5A). We therefore predicted that cells treated with CDK inhibitors would be protected from HIV-1 infection. We then evaluated to what extent these dephosphorylation events impacted infections of macrophages by HIV-1 (Figure 5B). Macrophages treated with Dasatinib were strongly protected from infection, as was previously reported [21,24]. The indicated CDK inhibitors showed reduced HIV-1 infection to varying degrees. Comparatively, treatment with the CDK1-specific inhibitor reduced HIV-1 infection by 83.75% (Figure 5B), and with the CDK4-specific inhibitor by 83.13% (Figure 5B), while the dual specificity inhibitors CDK1/2 inhibitor III and CDK4/6 inhibitor IV were less protective, reducing infection by 11.56% and 41.56%, respectively.

A: SAMHD1 phosphorylation in macrophages is reduced by dasatinib and by specific inhibitors of CDK1, 2, 4 and 6. B: Pre-treatment of  macrophages with dasatinib and specific inhibitors of CDK1, 2, 4, and 6 reduces HIV-1 infection.

Figure 5: A: SAMHD1 phosphorylation in macrophages is reduced by dasatinib and by specific inhibitors of CDK1, 2, 4 and 6. B: Pre-treatment of macrophages with dasatinib and specific inhibitors of CDK1, 2, 4, and 6 reduces HIV-1 infection.

CONCLUSIONS

Taking our data together with previously available literature [15,18,20,21,38], we propose that dasatinib directly inhibits the serine/threonine kinases CDK1, CDK2, CDK4, and CKD6, thus resulting in potent dephosphorylation of SAMHD1 via redundant pathways, conferring protection of MDMs from infection by HIV-1.

ACKNOWLEDGMENTS

This work was supported by the University of Utah Inflammation, Immunology, and Infectious Diseases (3i) Initiative’s Emerging Infectious Diseases Fellowship (recipient, ESCPW) and by a seed grant from the University of Utah Vice President for Research and the Immunology, Inflammation, and Infectious Disease Initiative (recipient, VP). VP and MC were supported by NIH GRANT 5R01-AI143567.

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Williams ESCP, Szaniawski MA, Martins LJ, Innis EA, Alcamí J, et al. (2022) Dasatinib: Effects on the Macrophage Phospho Proteome with a Focus on SAMHD1 and HIV-1 Infection. Clin Res HIV/AIDS 8(1): 1053.

Received : 13 Oct 2022
Accepted : 28 Nov 2022
Published : 30 Nov 2022
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JSM Neurosurgery and Spine
ISSN : 2373-9479
Launched : 2013
Journal of Liver and Clinical Research
ISSN : 2379-0830
Launched : 2014
Journal of Drug Design and Research
ISSN : 2379-089X
Launched : 2014
JSM Clinical Oncology and Research
ISSN : 2373-938X
Launched : 2013
JSM Bioinformatics, Genomics and Proteomics
ISSN : 2576-1102
Launched : 2014
JSM Chemistry
ISSN : 2334-1831
Launched : 2013
Journal of Trauma and Care
ISSN : 2573-1246
Launched : 2014
JSM Surgical Oncology and Research
ISSN : 2578-3688
Launched : 2016
Annals of Food Processing and Preservation
ISSN : 2573-1033
Launched : 2016
Journal of Radiology and Radiation Therapy
ISSN : 2333-7095
Launched : 2013
JSM Physical Medicine and Rehabilitation
ISSN : 2578-3572
Launched : 2016
Annals of Clinical Pathology
ISSN : 2373-9282
Launched : 2013
Annals of Cardiovascular Diseases
ISSN : 2641-7731
Launched : 2016
Journal of Behavior
ISSN : 2576-0076
Launched : 2016
Annals of Clinical and Experimental Metabolism
ISSN : 2572-2492
Launched : 2016
Clinical Research in Infectious Diseases
ISSN : 2379-0636
Launched : 2013
JSM Microbiology
ISSN : 2333-6455
Launched : 2013
Journal of Urology and Research
ISSN : 2379-951X
Launched : 2014
Journal of Family Medicine and Community Health
ISSN : 2379-0547
Launched : 2013
Annals of Pregnancy and Care
ISSN : 2578-336X
Launched : 2017
JSM Cell and Developmental Biology
ISSN : 2379-061X
Launched : 2013
Annals of Aquaculture and Research
ISSN : 2379-0881
Launched : 2014
Clinical Research in Pulmonology
ISSN : 2333-6625
Launched : 2013
Journal of Immunology and Clinical Research
ISSN : 2333-6714
Launched : 2013
Annals of Forensic Research and Analysis
ISSN : 2378-9476
Launched : 2014
JSM Biochemistry and Molecular Biology
ISSN : 2333-7109
Launched : 2013
Annals of Breast Cancer Research
ISSN : 2641-7685
Launched : 2016
Annals of Gerontology and Geriatric Research
ISSN : 2378-9409
Launched : 2014
Journal of Sleep Medicine and Disorders
ISSN : 2379-0822
Launched : 2014
JSM Burns and Trauma
ISSN : 2475-9406
Launched : 2016
Chemical Engineering and Process Techniques
ISSN : 2333-6633
Launched : 2013
Annals of Clinical Cytology and Pathology
ISSN : 2475-9430
Launched : 2014
JSM Allergy and Asthma
ISSN : 2573-1254
Launched : 2016
Journal of Neurological Disorders and Stroke
ISSN : 2334-2307
Launched : 2013
Annals of Sports Medicine and Research
ISSN : 2379-0571
Launched : 2014
JSM Sexual Medicine
ISSN : 2578-3718
Launched : 2016
Annals of Vascular Medicine and Research
ISSN : 2378-9344
Launched : 2014
JSM Biotechnology and Biomedical Engineering
ISSN : 2333-7117
Launched : 2013
Journal of Hematology and Transfusion
ISSN : 2333-6684
Launched : 2013
JSM Environmental Science and Ecology
ISSN : 2333-7141
Launched : 2013
Journal of Cardiology and Clinical Research
ISSN : 2333-6676
Launched : 2013
JSM Nanotechnology and Nanomedicine
ISSN : 2334-1815
Launched : 2013
Journal of Ear, Nose and Throat Disorders
ISSN : 2475-9473
Launched : 2016
JSM Ophthalmology
ISSN : 2333-6447
Launched : 2013
Journal of Pharmacology and Clinical Toxicology
ISSN : 2333-7079
Launched : 2013
Annals of Psychiatry and Mental Health
ISSN : 2374-0124
Launched : 2013
Medical Journal of Obstetrics and Gynecology
ISSN : 2333-6439
Launched : 2013
Annals of Pediatrics and Child Health
ISSN : 2373-9312
Launched : 2013
JSM Clinical Pharmaceutics
ISSN : 2379-9498
Launched : 2014
JSM Foot and Ankle
ISSN : 2475-9112
Launched : 2016
JSM Alzheimer's Disease and Related Dementia
ISSN : 2378-9565
Launched : 2014
Journal of Addiction Medicine and Therapy
ISSN : 2333-665X
Launched : 2013
Journal of Veterinary Medicine and Research
ISSN : 2378-931X
Launched : 2013
Annals of Public Health and Research
ISSN : 2378-9328
Launched : 2014
Annals of Orthopedics and Rheumatology
ISSN : 2373-9290
Launched : 2013
Journal of Clinical Nephrology and Research
ISSN : 2379-0652
Launched : 2014
Annals of Community Medicine and Practice
ISSN : 2475-9465
Launched : 2014
Annals of Biometrics and Biostatistics
ISSN : 2374-0116
Launched : 2013
JSM Clinical Case Reports
ISSN : 2373-9819
Launched : 2013
Journal of Cancer Biology and Research
ISSN : 2373-9436
Launched : 2013
Journal of Surgery and Transplantation Science
ISSN : 2379-0911
Launched : 2013
Journal of Dermatology and Clinical Research
ISSN : 2373-9371
Launched : 2013
JSM Gastroenterology and Hepatology
ISSN : 2373-9487
Launched : 2013
TEST Journal of Dentistry
ISSN : 1234-5678
Launched : 2014
Annals of Nursing and Practice
ISSN : 2379-9501
Launched : 2014
JSM Dentistry
ISSN : 2333-7133
Launched : 2013
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