Degradation Of CXCR4 Research Articles

MARVEL domain containing CMTM4 affects CXCR4 trafficking.

The MARVEL proteins CMTM4 and CMTM6 control PD-L1, thereby influencing tumor immunity. We found that defective zebrafish cmtm4 slowed the development of the posterior lateral line (pLL) by altering the Cxcr4b gradient across the pLL primordium (pLLP). Analysis in mammalian cells uncovered that CMTM4 interacted with CXCR4, altering its glycosylation pattern, but did not affect internalization or degradation of CXCR4 in the absence of its ligand CXCL12. Synchronized release of CXCR4 from the endoplasmic reticulum revealed that CMTM4 slowed CXCR4 trafficking from the endoplasmic reticulum to the plasma membrane without affecting overall cell surface expression. Altered CXCR4 trafficking reduced ligand-induced CXCR4 degradation and affected AKT but not ERK1/2 activation. CMTM4 expression, in contrast to that of CXCR4, correlated with the survival of patients with renal cell cancer in the TCGA cohort. Furthermore, we observed that cmtm4 depletion promotes the separation of cells from the pLLP cell cluster in zebrafish embryos. Collectively, our findings indicate that CMTM4 exerts general roles in the biosynthetic pathway of cell surface molecules and seems to affect CXCR4-dependent cell migration.

Deletion of Wild-type p53 Facilitates Bone Metastatic Function by Blocking the AIP4 Mediated Ligand-Induced Degradation of CXCR4.

Background: Management of patients with prostate cancer and bone metastatic disease remains a major clinical challenge. Loss or mutation of p53 has been identified to be involved in the tumor progression and metastasis. Nevertheless, direct evidence of a specific role for wild-type p53 (wt-p53) in bone metastasis and the mechanism by which this function is mediated in prostate cancer remain obscure. Methods: The expression and protein levels of wt-53, AIP4, and CXCR4 in prostate cancer cells and clinical specimens were assessed by real-time PCR, immunohistochemistry and western blot analysis. The role of wt-p53 in suppressing aggressive and metastatic tumor phenotypes was assessed using in vitro transwell chemotaxis, wound healing, and competitive colocalization assays. Furthermore, whether p53 deletion facilitates prostate cancer bone-metastatic capacity was explored using an in vivo bone-metastatic model. The mechanistic model of wt-p53 in regulating gene expression was further explored by a luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. Results: Our findings revealed that wt-p53 suppressed the prostate cancer cell migration rate, chemotaxis and attachment toward the osteoblasts in vitro. The bone-metastatic model showed that deletion of wt-p53 remarkably increased prostate cancer bone-metastatic capacity in vivo. Mechanistically, wt-p53 could induce the ligand-induced degradation of the chemokine receptor CXCR4 by transcriptionally upregulating the expression of ubiquitin ligase AIP4. Treatment with the CXCR4 inhibitor AMD3100 or transduction of the AIP4 plasmid abrogated the pro-bone metastasis effects of TP53 deletion. Conclusion: Wt-p53 suppresses the metastasis of prostate cancer cells to bones by regulating the CXCR4/CXCL12 activity in the tumor cells/bone marrow microenvironment interactions. Our findings suggest that targeting the wt-p53/AIP4/CXCR4 axis might be a promising therapeutic strategy to manage prostate cancer bone metastasis.

Characterization of a new WHIM syndrome mutant reveals mechanistic differences in regulation of the chemokine receptor CXCR4

WHIM syndrome is a rare immunodeficiency disorder that is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis. While several gain-of-function mutations that lead to C-terminal truncations, frame shifts and point mutations in the chemokine receptor CXCR4 have been identified in WHIM syndrome patients, the functional effect of these mutations are not fully understood. Here, we report on a new WHIM syndrome mutation that results in a frame shift within the codon for Ser339 (S339fs5) and compare the properties of S339fs5 with wild-type CXCR4 and a previously identified WHIM syndrome mutant, R334X. The S339fs5 and R334X mutants exhibited significantly increased signaling compared to wild-type CXCR4 including agonist-promoted calcium flux and extracellular-signal-regulated kinase activation. This increase is at least partially due to a significant decrease in agonist-promoted phosphorylation, β-arrestin binding, and endocytosis of S339fs5 and R334X compared with wild-type CXCR4. Interestingly, there were also significant differences in receptor degradation, with S339fs5 having a very high basal level of degradation compared with that of R334X and wild-type CXCR4. In contrast to wild-type CXCR4, both R334X and S339fs5 were largely insensitive to CXCL12-promoted degradation. Moreover, while basal and agonist-promoted degradation of wild-type CXCR4 was effectively inhibited by the CXCR4 antagonist TE-14016, this had no effect on the degradation of the WHIM mutants. Taken together, these studies identify a new WHIM syndrome mutant, CXCR4-S339fs5, which promotes enhanced signaling, reduced phosphorylation, β-arrestin binding and endocytosis, and a very high basal rate of degradation that is not protected by antagonist treatment.

Canonical (CD74/CD44) and Non-Canonical (CXCR2, 4 and 7) MIF Receptors Are Differentially Expressed in Rheumatoid Arthritis Patients Evaluated by DAS28-ESR.

Macrophage migration inhibitory factor (MIF) significantly contributes to rheumatoid arthritis (RA) pathogenesis. We aimed to evaluate the canonical (CD74/CD44) and non-canonical MIF receptors (CXCR2,4 and 7) expression and sCD74 to establish their association with RA clinical activity according to DAS28-ESR. Methodology: 101 RA patients with different clinical activities (remission (n = 27), low (n = 16), moderate (n = 35) and high (n = 23)) and 9 control subjects (CS) were included. Expression was evaluated by flow cytometry and levels of soluble CD74 (sCD74) by ELISA. Data analysis was performed with FlowJov10.0, STATAv12.0, and GraphPad Prism v7.0. Results: According to disease activity, CXCR7 expression (percentage of expression and mean fluorescence intensity (MFI)) was higher in granulocytes from patients in remission, while the expression of CXCR4 was higher in patients with high disease activity (p < 0.05). The expression of CD74 was higher in B cells (p < 0.05) and monocytes (p < 0.01) from patients in remission. Regarding sCD74 levels these were higher in patients with high disease activity when compared to those in remission (p <0.05). Conclusions: The results support the need for further study of the role of sCD74 as a soluble MIF decoy receptor, sequestering it to negatively regulate MIF signaling though its membrane receptors. The expression patterns of CXCR4 and CXCR7 show that the latter is a scavenger-type receptor that prevents endocytosis and even degradation of CXCR4 under inflammatory conditions.

GPRASP proteins are critical negative regulators of hematopoietic stem cell transplantation

AbstractHematopoietic stem cell (HSC) transplantation (HSCT) is often exploited to treat hematologic disease. Donor HSCs must survive, proliferate, and differentiate in the damaged environment of the reconstituting niche. Illuminating molecular mechanisms regulating the activity of transplanted HSCs will inform efforts to improve HSCT. Here, we report that G-protein–coupled receptor–associated sorting proteins (GPRASPs) function as negative regulators of HSCT. Silencing of Gprasp1 or Gprasp2 increased the survival, quiescence, migration, niche retention, and hematopoietic repopulating activity of hematopoietic stem and progenitor cells (HSPCs) posttransplant. We further show that GPRASP1 and GPRASP2 promote the degradation of CXCR4, a master regulator of HSC function during transplantation. CXCR4 accumulates in Gprasp-deficient HSPCs, boosting their function posttransplant. Thus, GPRASPs negatively regulate CXCR4 stability in HSCs. Our work reveals GPRASP proteins as negative regulators of HSCT and CXCR4 activity. Disruption of GPRASP/CXCR4 interactions could be exploited in the future to enhance the efficiency of HSCT.

3116 – GPRASP FAMILY MEMBERS AS NOVEL REGULATORS OF B-CELL DEVELOPMENT DURING NORMAL AND MALIGNANT HEMATOPOIESIS.

In our search for novel regulators of hematopoietic stem cell transplantation (HSCT), we recently reported Gprasp1 and Gprasp2 as negative regulators of HSCT. These genes encode proteins that are necessary for the stability, cellular trafficking and the proper degradation of CXCR4, a master regulator of hematopoiesis. Consequently, the absence of either Gprasp1 or Gprasp2 causes increased sensitivity to SDF-1 chemoattractant effect, enhancing HSCs repopulating activity after transplant. Strikingly, although Gprasp gene knockdown enhanced the repopulating activity of HSC at early time-points post-transplant, we observed that a significant number of mice transplanted with Gprasp-deficient hematopoietic progenitors developed an immature B-cell hematologic malignancy, typically at >20 weeks post-transplant. Since CXCR4 plays a crucial role in B-cell differentiation and, more specifically, in the movement of immature B-cells between the light and dark zones of the germinal center, we hypothesize that GPRASP family members are key regulators of CXCR4 during B-cell development during normal and malignant hematopoiesis. Thus, we followed mice transplanted with hematopoietic progenitors treated with control, Gprasp1 or Gprasp2-shRNAs for up to one year post-transplant. The onset of B-cell malignancy coincided with an accumulation of bone marrow common lymphoid progenitors, a complete disruption of the immunophenotype of B-cell progenitors and a loss of mature B-cells in the peripheral blood. Transformed cells invaded bone marrow, spleen, and lymph nodes in primary recipients. 100% of secondary recipients of leukemic cells developed a phenotypically identical malignancy <6 weeks post-transplant. As the dark zone of the geminal center is a region where somatic hypermutation is naturally exacerbated, we hypothesize that Gprasp-deficient B-cell progenitors are arrested in an immature state in the germinal center, increasing the likelihood of malignant transformation.

3117 – GPRASP2 REGULATES CXCR4 STABILITY AND SIGNAL TRANSDUCTION IN HUMAN HEMATOPOIETIC CELLS WITH IMPLICATIONS FOR HSPC TRANSPLANTATION

Transplantation of hematopoietic stem and progenitor cells (HSPCs) is a life-saving therapy that is limited by donor availability and low cell numbers in sources such as cord blood. Advances in our understanding of the biochemical pathways that determine HSPC engraftment will allow for the optimization of transplantation strategies. We previously found that knockdown of GPRASP (G-protein Coupled Receptor Associated Sorting Protein) protein family members increases CXCR4 abundance and transplantation efficiency in murine HSPCs. Here, we demonstrate that GPRASP2 facilitates the acute agonist-induced degradation of CXCR4 in human hematopoietic cells and limits signaling downstream of CXCR4. Initial data also indicate that loss of GPRASP2 may increase the transplantation efficiency of human CD34+ cells. An antibody protection assay performed in HL60 cells demonstrated that knockdown of GPRASP2 significantly increases stability of CXCR4 following SDF-1 treatment. Phosphoflow experiments were performed to quantify the impact of GPRASP2 on signaling pathways downstream of CXCR4 including AKT, ERK1/2, p38, and JAK/STAT. SDF-1 treatment primarily resulted in phosphorylation of ERK1/2 and this was significantly increased by GPRASP2 knockdown. To test the role of GPRASP2 in human HSPC transplantation, CD34+ cells were edited by CRISPR/Cas9 with sgRNAs targeting GPRASP2 or the AAVS1 safe harbor site and then xenotransplanted into NBSGW mice. Initial results indicate that GPRASP2 KO results in higher human chimerism than is seen for AAVS1-targeted cells. In addition to assessing chimerism between transplants, changes in INDEL frequency within each transplant will be tracked for 16 weeks to determine the fitness of GPRASP2 knockout cells as compared to non-edited cells. This work reveals novel aspects of CXCR4 regulation by GPRASP2 and illuminates a potential point of intervention for the optimization of HSPC transplantation.

Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans.

Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.

Impaired migration capacity in monocytes derived from patients with Gaucher disease.

Gaucher disease (GD) is characterized by glucocerebroside (GC) accumulation due to defective activity of the glucocerebrosidase (GlcCerase) enzyme. Monocytes and macrophages exhibit the highest GlcCerase activity and are most prominently affected by GC engorgement. As GD patients tend to exert various immune system-related changes, this study was designed to investigate potential effects of monocyte dysfunction on these alterations. Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) of untreated GD patients and healthy volunteers. Monocyte migration capacity towards SDF1α was assessed. The GD patients exhibited reduced numbers of monocytes and decreased capability of SDF1α-dependent monocyte migration. Evaluation of CXCR4, the SDF1α receptor, revealed reduced expression of surface CXCR4 on GD-derived monocytes, despite similar CXCR4 mRNA transcript levels in the monocytes of healthy volunteers and GD patients. Reduction of surface CXCR4 was accompanied by increased intracellular CXCR4 levels in patient monocytes. This elevated intracellular CXCR4 might reflect significantly increased SDF1α concentrations characterizing patients' serum and the lysosomal impairment of GD, resulting in decreased degradation of CXCR4. Different distributions of CXCR4 expression observed in the two groups explain impaired SDF1α-dependent monocyte migration. Reduced numbers and impaired migration capacity of GD-derived monocytes could contribute to abnormal inflammation and GD-associated immune alterations seen in these patients.

CXCR4 blockade induces atherosclerosis by affecting neutrophil function

AimsThe SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. Methods and resultsImmunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4+ cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr−/− mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. ConclusionIn conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.

Novel Roles for the E3 Ubiquitin Ligase Atrophin-interacting Protein 4 and Signal Transduction Adaptor Molecule 1 in G Protein-coupled Receptor Signaling

The CXCL12/CXCR4 signaling axis plays an important role in human health and disease; however, the molecular mechanisms mediating CXCR4 signaling remain poorly understood. Ubiquitin modification of CXCR4 by the E3 ubiquitin ligase AIP4 is required for lysosomal sorting and degradation, which is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. CXCR4 sorting is regulated by an interaction between endosomal localized arrestin-2 and STAM-1, an ESCRT-0 component. Here, we report a novel role for AIP4 and STAM-1 in regulation of CXCR4 signaling that is distinct from their function in CXCR4 trafficking. Depletion of AIP4 and STAM-1 by siRNA caused significant inhibition of CXCR4-induced ERK-1/2 activation, whereas overexpression of these proteins enhanced CXCR4 signaling. We further show that AIP4 and STAM-1 physically interact and that the proline-rich region in AIP4 and the SH3 domain in STAM-1 are essential for the interaction. Overexpression of an AIP4 catalytically inactive mutant and a mutant that shows poor binding to STAM-1 fails to enhance CXCR4-induced ERK-1/2 signaling, as compared with wild-type AIP4, suggesting that the interaction between AIP4 and STAM-1 and the ligase activity of AIP4 are essential for ERK-1/2 activation. Remarkably, a discrete subpopulation of AIP4 and STAM-1 resides in caveolar microdomains with CXCR4 and appears to mediate ERK-1/2 signaling. We propose that AIP4-mediated ubiquitination of STAM-1 in caveolae coordinates activation of ERK-1/2 signaling. Thus, our study reveals a novel function for ubiquitin in the regulation of CXCR4 signaling, which may be broadly applicable to other G protein-coupled receptors.

Arrestin-2 Interacts with the Endosomal Sorting Complex Required for Transport Machinery to Modulate Endosomal Sorting of CXCR4

The chemokine receptor CXCR4, a G protein-coupled receptor, is targeted for lysosomal degradation via a ubiquitin-dependent mechanism that involves the endosomal sorting complex required for transport (ESCRT) machinery. We have reported recently that arrestin-2 also targets CXCR4 for lysosomal degradation; however, the molecular mechanisms by which this occurs remain poorly understood. Here, we show that arrestin-2 interacts with ESCRT-0, a protein complex that recognizes and sorts ubiquitinated cargo into the degradative pathway. Signal-transducing adaptor molecule (STAM)-1, but not related STAM-2, interacts directly with arrestin-2 and colocalizes with CXCR4 on early endosomal antigen 1-positive early endosomes. Depletion of STAM-1 by RNA interference and disruption of the arrestin-2/STAM-1 interaction accelerates agonist promoted degradation of CXCR4, suggesting that STAM-1 via its interaction with arrestin-2 negatively regulates CXCR4 endosomal sorting. Interestingly, disruption of this interaction blocks agonist promoted ubiquitination of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) but not CXCR4 and STAM-1 ubiquitination. Our data suggest a mechanism whereby arrestin-2 via its interaction with STAM-1 modulates CXCR4 sorting by regulating the ubiquitination status of HRS.

The E3 Ubiquitin Ligase Atrophin Interacting Protein 4 Binds Directly To The Chemokine Receptor CXCR4 Via a Novel WW Domain-mediated Interaction

The E3 ubiquitin ligase atrophin interacting protein 4 (AIP4) mediates ubiquitination and down-regulation of the chemokine receptor CXCR4. AIP4 belongs to the Nedd4-like homologous to E6-AP carboxy terminus domain family of E3 ubiquitin ligases, which typically bind proline-rich motifs within target proteins via the WW domains. The intracellular domains of CXCR4 lack canonical WW domain binding motifs; thus, whether AIP4 is targeted to CXCR4 directly or indirectly via an adaptor protein remains unknown. Here, we show that AIP4 can interact directly with CXCR4 via a novel noncanonical WW domain-mediated interaction involving serine residues 324 and 325 within the carboxy-terminal tail of CXCR4. These serine residues are critical for mediating agonist-promoted binding of AIP4 and subsequent ubiquitination and degradation of CXCR4. These residues are phosphorylated upon agonist activation and phosphomimetic mutants show enhanced binding to AIP4, suggesting a mechanism whereby phosphorylation mediates the interaction between CXCR4 and AIP4. Our data reveal a novel noncanonical WW domain-mediated interaction involving phosphorylated serine residues in the absence of any proline residues and suggest a novel mechanism whereby an E3 ubiquitin ligase is targeted directly to an activated G protein-coupled receptor.

CXCR4 Expression and Biological Activity Is Dependent on Oxygen Partial Pressure in Acute Myeloid Leukemia.

CXCR4, the receptor for bone marrow stroma derived SDF-1, has recently been studied in normal hematopoiesis and hematologic malignancies. Increased expression of CXCR4 by leukemic blasts has been reported by us and others (Konoplev S. et al, Cancer 2007) to be associated with poor prognosis in acute myeloid leukemia (AML). However, all in-vitro studies are usually carried out under unphysiological, i.e. normoxic (21% O2) conditions. We hypothesized that the pO2 in vitro has major impact on the expression of CXCR4, a key receptor for cell migration and intracellular signalling. Thus, pO2 of bone marrow aspirates was measured using the i-STAT Portable Clinical Analyzer and a hypoxic workstation was used providing constant low oxygen content. Surface and total CXCR4 expression was examined in leukemic cell lines and patient samples by flow cytometry, confocal microscopy and Western blotting (WB). In 19 patients, the median pO2 of the bone marrow was determined as 46.1±12.8 mmHg (6.1±1.7%) with no significant difference between patients with AML (n=7, pO2 41.3±11.2 mmHg) and patients in CR (n=12, pO2 48.3±15.9 mmHg). This level of hypoxia significantly increases surface and total expression of CXCR4 in the leukemic cell lines U937 and OCI-AML3 as well as in samples from patients with AML, as compared to normoxic conditions (~2.8fold increase). This increase happened mainly within the first 2–8 hours of hypoxia and was unrelated to increased CXCR4 transcription, as shown by PCR. Re-oxygenation of leukemic cells resulted in a statistical significant degradation of CXCR4 (~3fold decrease) in all examined cell lines and patient samples (n=10). This loss of CXCR4 is very rapid (within 5 minutes of re-oxygenation) and was detected by flow cytometry, confocal microscopy and WB. This phenomenon was independent of proteasome activity and ATP. Detailed analysis of membraneous lipid rafts by sucrose density separation, cholesterol depletion and flow cytometry analysis for GM1 gangliosides showed structural (distinct re-distribution of Lck in lipid rafts) and quantitative changes (loss of cholesterol and CXCR4) during re-oxygenation. Moreover, part of the loss of CXCR4 can be attributed to sequestration of microparticles into the extracellular environment as shown by WB of supernatant of re-oxygenated cells and by a significant increase (~1.5fold) in the amount of microparticles released by cells (cell lines U937 and OCI-AML3 and additional patient samples) during the process of re-oxygenation, as measured by flow cytometry. In summary, this study determined the oxygen content of CR and leukemic bone marrow samples as 6.1±1.7%. This pO2 is associated with an increase in CXCR4 expression on AML cells, while re-oxygenation leads to a rapid decrease of CXCR4, perhaps in part by shedding of CXCR4- containing microparticles. These studies point to the importance of studying leukemic blasts under physiologic, i.e. hypoxic conditions.

Effect of pan-histone deacetylase inhibitor panobinostat (LBH589) on CXCR4 levels and signaling and on anti-leukemia activity in combination with CXCR4 antagonists

14541 Background: Bone marrow stroma-derived factor-1 (SDF-1, CXCL12) binds and activates the chemokine receptor CXCR4, which regulates the trafficking and mobilization of normal and leukemic hematopoietic cells. CXCR4 signaling thorough AKT and c-Raf also promotes survival and decreases sensitivity of AML cells to anti-leukemia agents. Both CXCL12 and CXCR4 are overexpressed and confer poor prognosis in AML. Treatment with the CXCR4 antagonist AMD3100 sensitizes AML cells to anti-leukemia drugs. We determined the effect of panobinostat (P) and/or FC-131 (a new generation CXCR4 inverse agonist) on CXCR4 expression and signaling in cultured and primary AML cells. Methods: Following treatment of human AML OCI-AML3, HL-60, and Jurkat cells, as well as primary AML cells with P (10 to 50 nM) and/or FC-131 (10 nM) with our without 10 nM of CXCL12, cell surface expression of CXCR4 and apoptosis (Annexin V/PI staining) were determined by flow cytometry. Intracellular protein levels of CXCR4, p-AKT, p-ERK1/2, heat shock protein (hsp) 70 and hsp90 were determined by immunoblot (IB) analyses. Binding of CXCR4 to hsp90 and hsp70, as well as CXCR4 acetylation, were determined by immunoprecipitation (IP)/IB analyses. Results: Treatment with P depleted the intracellular and surface expression of CXCR4 in the presence or absence of CXCL12. P induced acetylation of hsp90 and decreased its binding to CXCR4. P also induced acetylation and increased membrane binding of CXCR4 to hsp70. P promoted degradation of CXCR4 by the proteasome. Treatment with P alone also decreased p-AKT and p-ERK1/2. Co- treatment with P markedly attenuated the phosphorylation (p) of AKT and ERK1/2 induced by CXCL12, casuing apoptosis of up to 50% of cultured and primary AML cells. Co-treatment with P and FC-131 caused greater depletion of CXCR4, p-AKT and p-ERK1/2 levels, and synergistically induced apoptosis of the cultured AML cells (combination indices of < 1.0, utilizing isobologram analysis). Conclusions: CXCR4 is chaperoned by hsp90, and treatment with P depletes CXCR4 levels and signaling in AML cells. The novel combination of FC-131 and P is synergistically active and its in vivo ant-AML efficacy is currently under evaluation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Novartis Institute for Biomedical Research Inc. Novartis Institute for Biomedical Research Inc. Merck & Co., Novartis, Novartis Institute for Biomedical Research Inc.

Arrestin-2 Interacts with the Ubiquitin-Protein Isopeptide Ligase Atrophin-interacting Protein 4 and Mediates Endosomal Sorting of the Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is rapidly targeted for lysosomal degradation by the E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4). Although it is known that AIP4 mediates ubiquitination and degradation of CXCR4 and that perturbations in these events contribute to disease, the mechanisms mediating AIP4-dependent regulation of CXCR4 degradation remain poorly understood. Here we show that AIP4 directly interacts with the amino-terminal half of nonvisual arrestin-2 via its WW domains. We show that depletion of arrestin-2 by small interfering RNA blocks agonist-promoted degradation of CXCR4 by preventing CXCR4 trafficking from early endosomes to lysosomes. Surprisingly, CXCR4 internalization and ubiquitination remain intact, suggesting that the interaction between arrestin-2 and AIP4 is not required for ubiquitination of the receptor at the plasma membrane but perhaps for a later post-internalization event. Accordingly, we show that activation of CXCR4 promotes the interaction between AIP4 and arrestin-2 that is consistent with a time when AIP4 co-localizes with arrestin-2 on endocytic vesicles. Taken together, our data suggest that the AIP4.arrestin-2 complex functions on endosomes to regulate sorting of CXCR4 into the degradative pathway.

Pan-Histone Deacetylase (HDAC) Inhibitor LBH589 Depletes CXCR4 Levels and Signaling, Exerting Synergistic Anti-Leukemia Activity in Combination with CXCR4 Antagonists.

The trafficking and mobilization of normal hematopoietic progenitors and their leukemic counterparts is programmed in part by chemotactic gradients of CXCL12, which are transduced by CXCR4. This mechanism also results in decreased sensitivity to pro-apoptotic and anti-leukemic agents, mediated through CXCR4 activation of Akt and Raf phosphorylation and/or harboring in a microenvironmental niche. Both CXCL12 and CXCR4 are overexpressed in AML and expression of CXCR4 has been associated with a poor prognosis. Moreover, inhibition of CXCR4 with AMD3100 enhanced the sensitivity of leukemic myeloblasts to anti-leukemic agents. We therefore explored therapeutic mechanisms to decrease CXCR4 expression and tested them in combination with AMD3100 as well as a new generation CXCR4 inverse agonist, FC131. Exposure to 10 to 50 nM of the pan-HDAC inhibitor LBH589 (Novartis) depleted mRNA and protein levels of CXCR4 in the cultured human acute leukemia OCI-AML3, HL-60 and Jurkat cells, as well as in primary AML cells in a dose and time-dependent manner. LBH589 depleted CXCR4 levels in the presence or absence of 10 nM of CXCL12 in the culture medium. LBH589 mediated depletion of CXCR4 levels was partly due to decreased CXCR4 mRNA levels (by RT-PCR analysis). LBH589 induced acetylation of heat shock protein (hsp) 90 and attenuated the binding of CXCR4 to hsp90 with subsequent degradation of CXCR4 by the proteasome. LBH589 treatment also increased hsp70 levels and acetylation, as well as its binding to CXCR4, which also resulted in increased extra-cellular, cell surface co-localization of CXCR4 and hsp70. This was markedly inhibited by siRNA-mediated knockdown of hsp70 in HL-60 cells. While exposure of cultured and primary AML cells to CXCL12 markedly increased cytosolic levels of p-AKT and p-ERK1/2, co-treatment with LBH589 markedly attenuated the phosphorylation of AKT and ERK1/2 induced by CXCL12, resulting in apoptosis of up to 50% of cultured and primary AML cells. Treatment with AMD3100 (10 uM for 24 hours) alone decreased levels of CXCR4, p-AKT and p-ERK1/2, without significantly increasing apoptosis of AML cells. Notably, co-treatment with LBH589 and AMD3100 caused greater depletion of CXCR4, p-AKT and p-ERK1/2 levels, and exerted synergistic apoptotic effects against AML cells with combination indices of < 1.0 utilizing isobologram and median effect analyses. Co-treatment with LBH589 and FC131 (10 nM for 24 hours), which is a more potent CXCR4 antagonist than AMD3100, also induced synergistic apoptosis of cultured and primary AML cells. Taken together, these findings provide direct evidence that CXCR4 is a novel target depleted by LBH589 in AML cells. Furthermore, our in vitro findings highlight the novel combination of LBH589 and a CXCR4 antagonist, AMD3100 or FC131, exerts a synergistic effect on acute leukemia cells. These findings strongly support the in vivo testing of this synergistic combination in the therapy of human acute leukemias that express CXCR4.

Limiting Carryover Events Resulting from Cell Surface Retention of VSV-G Pseudotyped HIV-Lentivector Particles on Hematopoietic Targets

Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.

CISK attenuates degradation of the chemokine receptor CXCR4 via the ubiquitin ligase AIP4

HER2 overexpression in cancers causes hyperactivation of the PI 3-kinase pathway and elevated levels of the chemokine receptor CXCR4, which is strongly associated with increased metastatic potential. Here, we provide evidence that the cytokine-independent survival kinase CISK is activated downstream of the PI 3-kinase-dependent kinase PDK1 on endosomes and negatively regulates the lysosomal degradation of CXCR4. We demonstrate that CISK prevents CXCR4 degradation by inhibiting sorting of the receptor from early endosomes to lysosomes. In contrast, CISK does not interfere with ligand-induced degradation of epidermal growth factor receptors. CISK strongly interacts and colocalizes with the E3 ubiquitin ligase AIP4, which is important for the ubiquitin-dependent lysosomal degradation of CXCR4. Moreover, the observed inhibition is both dependent on the interaction between CISK and AIP4 and on the activation status of CISK. Consistent with this, an activated form of CISK but not of the related kinase SGK1 phosphorylates specific sites of AIP4 in vitro. Taken together, these results reveal a critical function of CISK in specifically attenuating ubiquitin-dependent degradation of CXCR4, and provide a mechanistic link between the PI 3-kinase pathway and CXCR4 stability.