Surface Expression Of CXCR4 Research Articles

Pulse Activation of Retinoic Acid Receptor Enhances Hematopoietic Stem Cell Homing by Controlling CXCR4 Membrane Presentation.

The interplay between metabolic signaling and stem cell biology has gained increasing attention, though the underlying molecular mechanisms remain incompletely elucidated. In this study, we identify and characterize the role of adapalene (ADA), a retinoic acid receptor (RAR) agonist, in modulating the migration behavior of hematopoietic stem cells (HSCs). Our initial findings reveal that ADA treatment suppresses hematopoietic stem and progenitor cell (HSPC) mobilization induced by AMD3100 and G-CSF. Furthermore, we demonstrate that ADA treatment upregulates the surface expression of CXCR4 on HSPCs, resulting in enhanced chemotaxis towards CXCL12. Mechanistically, our study suggests that ADA enhances CXCR4 surface presentation without increasing CXCR4 mRNA levels, pointing towards a non-canonical role of RAR signaling in regulating intracellular trafficking of CXCR4. In vivo experiments show that ADA administration significantly enhances HSC homing efficiency. Additionally, competitive transplantation assays indicate a marked increase in donor chimerism following ADA treatment. These findings highlight the critical role of retinoic acid signaling in regulating HSC homing and suggest its potential for advancing novel HSC-based therapeutic strategies.

Pim Kinase Inhibition Disrupts CXCR4 Signalling in Megakaryocytes and Platelets by Reducing Receptor Availability at the Surface.

A key step in platelet production is the migration of megakaryocytes to the vascular sinusoids within the bone marrow. This homing is mediated by the chemokine CXCL12 and its receptor CXCR4. CXCR4 is also a positive regulator of platelet activation and thrombosis. Pim-1 kinase has been shown to regulate CXCR4 signalling in other cell types, and we have previously described how Pim kinase inhibitors attenuate platelet aggregation to CXCL12. However, the mechanism by which Pim-1 regulates CXCR4 signalling in platelets and megakaryocytes has yet to be elucidated. Using human platelets, murine bone marrow-derived megakaryocytes, and the megakaryocyte cell line MEG-01, we demonstrate that pharmacological Pim kinase inhibition leads to reduced megakaryocyte and platelet function responses to CXCL12, including reduced megakaryocyte migration and platelet granule secretion. Attenuation of CXCL12 signalling was found to be attributed to the reduced surface expression of CXCR4. The decrease in CXCR4 surface levels was found to be mediated by rapid receptor internalisation, in the absence of agonist stimulation. We demonstrate that pharmacological Pim kinase inhibition disrupts megakaryocyte and platelet function by reducing constitutive CXCR4 surface expression, decreasing the number of receptors available for agonist stimulation and signalling. These findings have implications for the development and use of Pim kinase inhibitors for the treatment of conditions associated with elevated circulating levels of CXCL12/SDF1α and increased thrombotic risk.

CD74 is a functional MIF receptor on activated CD4+ T cells

Next to its classical role in MHC II-mediated antigen presentation, CD74 was identified as a high-affinity receptor for macrophage migration inhibitory factor (MIF), a pleiotropic cytokine and major determinant of various acute and chronic inflammatory conditions, cardiovascular diseases and cancer. Recent evidence suggests that CD74 is expressed in T cells, but the functional relevance of this observation is poorly understood. Here, we characterized the regulation of CD74 expression and that of the MIF chemokine receptors during activation of human CD4+ T cells and studied links to MIF-induced T-cell migration, function, and COVID-19 disease stage. MIF receptor profiling of resting primary human CD4+ T cells via flow cytometry revealed high surface expression of CXCR4, while CD74, CXCR2 and ACKR3/CXCR7 were not measurably expressed. However, CD4+ T cells constitutively expressed CD74 intracellularly, which upon T-cell activation was significantly upregulated, post-translationally modified by chondroitin sulfate and could be detected on the cell surface, as determined by flow cytometry, Western blot, immunohistochemistry, and re-analysis of available RNA-sequencing and proteomic data sets. Applying 3D-matrix-based live cell-imaging and receptor pathway-specific inhibitors, we determined a causal involvement of CD74 and CXCR4 in MIF-induced CD4+ T-cell migration. Mechanistically, proximity ligation assay visualized CD74/CXCR4 heterocomplexes on activated CD4+ T cells, which were significantly diminished after MIF treatment, pointing towards a MIF-mediated internalization process. Lastly, in a cohort of 30 COVID-19 patients, CD74 surface expression was found to be significantly upregulated on CD4+ and CD8+ T cells in patients with severe compared to patients with only mild disease course. Together, our study characterizes the MIF receptor network in the course of T-cell activation and reveals CD74 as a novel functional MIF receptor and MHC II-independent activation marker of primary human CD4+ T cells.

Fine-tuning spatial-temporal dynamics and surface receptor expression support plasma cell-intrinsic longevity

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

Fine-tuning spatial-temporal dynamics and surface receptor expression support plasma cell-intrinsic longevity.

Durable serological memory following vaccination is critically dependent on the production and survival of long-lived plasma cells (LLPCs). Yet, the factors that control LLPC specification and survival remain poorly resolved. Using intravital two-photon imaging, we find that in contrast to most plasma cells (PCs) in the bone marrow (BM), LLPCs are uniquely sessile and organized into clusters that are dependent on APRIL, an important survival factor. Using deep, bulk RNA sequencing, and surface protein flow-based phenotyping, we find that LLPCs express a unique transcriptome and phenotype compared to bulk PCs, fine-tuning expression of key cell surface molecules, CD93, CD81, CXCR4, CD326, CD44, and CD48, important for adhesion and homing. Conditional deletion of Cxcr4 in PCs following immunization leads to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and ultimately accelerated decay of antibody titer. In naïve mice, the endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting LLPC specification is non-random. As mice age, the BM PC compartment becomes enriched in LLPCs, which may outcompete and limit entry of new PCs into the LLPC niche and pool.

Abstract 732: Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia

Abstract Introduction: CXCR4 is a chemokine receptor for CXCL12 that plays a critical role in homing of hematopoietic stem cells (HSCs) to the bone marrow microenvironment. The CXCR4 inhibitor plerixafor is used to increase mobilization of peripheral blood stem cells in combination with filgrastim. This pathway has also been investigated for applications in hematologic malignancies such as acute myeloid leukemia (AML), including clinical trials, with the goal of disrupting AML from the protective marrow environment. These have included use of plerixafor with 7+3 or MEC, BL-8040 with cytarabine, and ulocuplumab with MEC. For the ulocuplumab trial, at the study site of the authors, CR/CRi correlated with >10% CXCR4 expression (Becker et al. ASH 2014). The objective of this preclinical study is to identify the AML patient population most likely to respond to CXCR4 inhibition, and the role of combined beta-adrenergic blockade, as the latter has been shown pre-clinically to augment mobilization of HSCs when combined with a CXCR4 inhibitor, GPC-100 (Sukhtankar et al. PLoS One, 2023). Procedures: AML patient samples (N=124 including 90 blood and 34 bone marrow samples, and 21 with both) were obtained with informed consent on an IRB approved protocol. Multicolor flow cytometry was performed with sequential gating on blasts (by FSC/SSC, CD45/SSC, CD34 or phenotype), and leukemia stem cells (LSCs:CD34+CD38−,CD123+), then analysis for CXCR4 and beta-2 adrenergic receptor (ADRB2) surface expression. Correlations between CXCR4 expression and clinical characteristics including age, gender, new diagnosis vs. relapse, ELN2022 risk, cytogenetics, FLT3/NPM1 mutation status, CR1 duration, and overall survival were performed. GraphPad Prism9 was used for statistical analysis, and Mann Whitney test (two groups) and ordinary one-way ANOVA (>2 groups) were used for comparisons. Results: The range of CXCR4 expression by AML blasts is 0.05 to 99.7% (median 13.1%; mean 16.2%), and by AML LSCs 0 to 99% (median 2%, mean 15.7%). The expression by blasts and LSCs for each patient exhibited tight correlation, R2=0.88 p=1.23 × 10e-25). There was higher % expression of CXCR4 by both blasts and LSCs for new diagnosis, as compared to relapsed, patient samples (p=0.002). There was no correlation between level of CXCR4 expression and the other clinical features. For a subset of 20 patient samples analyzed for both CXCR4 and ADBR2 expression by AML blasts, ADBR2 expression ranged from 0.4 to 20.1%. For the samples that express ADBR2, there was high level expression of CXCR4, range 35.9 to 98.7% (median 78.4%, mean 76%). Conclusion: These studies support further investigation of whether simultaneous blockade of CXCR4 and ADBR2 may potentiate chemotherapy response in AML by limiting microenvironment mediated chemotherapy protection and reveal that patients with new diagnosis may be more susceptible to this approach. Citation Format: Brenda Loera, Chunxiao Zhang, Pina M. Cardarelli, Niña G. Caculitan, Sylvia Chien, Jin Dai, Vivian G. Oehler, Jianying Zhang, Guido Marcucci, Pamela S. Becker. Toward optimizing CXCR4 inhibition with beta adrenergic blockade to enhance chemotherapy response in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 732.

General anaesthetics reduce acute lymphoblastic leukaemia malignancies in vitro and in vivovia CXCR4 and osteopontin mediated mechanisms.

Acute lymphoblastic leukaemia (ALL) is a common type of cancer in children. General anaesthetics are often used on patients undergoing painful procedures during ALL treatments but their effects on ALL malignancy remain unknown. Herein, we aim to study the effect of propofol and sevoflurane on the migration, homing and chemoresistance of ALL cells. NALM-6 and Reh cells were treated with propofol (5 and 10 μg/ml) or sevoflurane (3.6%) in vitro for six hours. Then, cells were harvested for adhesion assay and migration assay in vitro. In in vivo experiments, GFP-NALM-6 cells were pre-treated with propofol (10 μg/ml) or sevoflurane (3.6%) for six hours. Then, cells were injected intravenously to C57BL/6 female mice followed by intravital microscopy. For chemoresistance study, cells were treated with rising concentrations of Ara-c (0.05-50 nM) plus 10μg/ml of propofol or Ara-C plus 3.6% of sevoflurane for 4 hours, followed by the assessment of cell viability via CCK-8 assay and detection of autophagy via flow cytometry. Both anaesthetics reduced in vivo migration and in vivo homing as exemplified by 1) the reduction in the number of cells entering the bone marrow and 2) the disturbance in homing location in relation to endosteal surface. Our results indicated that general anaesthetics reduced the surface CXCR4 expression and the adhesion of leukaemia cells to thrombin cleaved osteopontin (OPN) was reduced. Those changes might result in the alterations in migration and homing. In addition, both anaesthetics sensitised ALL cells to Ara-c possibly through CXCR4 mediated mechanisms. Propofol but not sevoflurane enhanced chemo-related cell death via inducing cytotoxic autophagy. Together, our data suggest that both propofol and sevoflurane could reduce ALL migration, and homing in vivo and in vitro via CXCR4 and OPN mediated mechanisms. Both anaesthetics could sensitise ALL cells to chemotherapy possibly via CXCR4 mediated mechanisms.

Altered Cellular Dynamics and Surface Expression Promote Long-Lived Plasma Cell Survival

Serological memory following vaccination decline with aging, indicating reduced survival of new plasma cells (PCs). We report that with age, bone marrow (BM) PC pool is enriched in long-lived plasma cells (LLPCs), leading to reduced PC turnover, suggesting increased competition. Using in vivo imaging, we find that LLPCs are highly organized into April-dependent clusters. Based on bulk RNA sequencing, and flow-based surface phenotyping, LLPCs exhibit unique transcriptome and proteome, fine tuning expression of key cell surface molecules, including CD93, CD81, CXCR4, CD326, CD44 and CD48, important for adhesion and homing. To test their functional role, Cxcr4 was conditionally deleted in PCs following immunization, which led to rapid mobilization from the BM, reduced survival of antigen-specific PCs, and accelerated decay of antibody titer. Analysis of endogenous LLPCs BCR repertoire exhibits reduced diversity, reduced somatic mutations, and increased public clones and IgM isotypes, particularly in young mice, suggesting non-random selection into the LLPC pool that is age-dependent.

Chronic treatment with TNF-α, alone and in combination with Takinib, SB203580 and metformin induce cell death in breast cancer

Breast cancer (BC) is the most common malignancy, and the largest cause of cancer death among women. The interactions between tumor cells and tumor micro environmental factors have a major impact on tumor progression. One of the critical pro-inflammatory cytokines present in breast cancer tumor microenvironment is TNF-α. The aim of this study was to evaluate the long-term effect of TNF-α (1 week) along with p38 or TAK1 inhibitors as well as metformin on induction of cellular death, cancer stem cell and expression of metastatic marker CXCR4. MCF-7 and MDA-MB-231 cells were treated with TNF-α for one week and then were treated with combination of Takinib, SB203580 or Metformin; after all treatments were done, cell proliferation, cellular death, surface expression of CXCR4, CD44 and CD24 were determined. The results showed that treatment with TNF-α alone or in combination with Takinib, SB203580 and metformin elevated induction of cellular death in both cell lines compared to the control group. TNF-α also increased CXCR4 expression in MCF-7 cells, but it reduced its expression in the MDA-MB-231 cells. Also, breast cancer stem cells (BCSCs) population decreased in MDA-MB-231 cells treated with TNF-α alone or in combination with SB203580 and metformin. Although, in MCF-7 cells only combination of TNF-α and Takinib reduced BCSCs population in a time dependent manner. Altogether, we showed that TNF-α alone or in combination with other treatments can affect the progression of breast cancer.

The interplay of FLT3 and CXCR4 in acute myeloid leukemia: an ongoing debate.

FLT3 mutations are very frequent in AML and utilization of FLT3 inhibitors as approved treatment options are very common. Despite the initial success of inhibitor treatment, the development of resistances against this treatment is a major challenge in AML therapy. One of the mechanisms causing resistance is the homing of the leukemic cells in the protective niche of the bone marrow microenvironment (BMM). A pathway mediating homing to the BMM and leukemic cell survival is the CXCL12/CXCR4 axis. The analysis of patient samples in several independent studies indicated that FLT3-ITD expression led to higher CXCR4 surface expression. However, several in vitro studies reported contradictory findings, suggesting that FLT3-ITD signaling negatively influenced CXCR4 expression. In this commentary, we provide an overview summarizing the studies dealing with the relationship of FLT3 and CXCR4. Taken together, the current research status is not sufficient to answer the question whether FLT3 and CXCR4 act together or independently in leukemia progression. Systematic analyses in model cell systems are needed to understand the interplay between FLT3 and CXCR4, since this knowledge could lead to the development of more effective treatment strategies for AML patients.

Effects of bromodomain and extra-terminal inhibitor JQ1 and interleukin-6 on breast cancer cells.

Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

Nuclear Tubulin Enhances CXCR4 Transcription and Promotes Chemotaxis Through TCF12 Transcription Factor in human Hematopoietic Stem Cells.

Tubulins are cytoskeleton components in all eukaryotic cells and play crucial roles in various cellular activities by polymerizing into dynamic microtubules. A subpopulation of tubulin has been shown to localize in the nucleus, however, the function of nuclear tubulin remains largely unexplored. Here we report that microtubule depolymerization specifically upregulates surface CXCR4 expression in human hematopoietic stem cells (HSCs). Mechanistically, microtubule depolymerization results in accumulation of tubulin subunits in the nucleus, leading to elevated CXCR4 transcription and increased chemotaxis of human HSCs. Treatment with microtubule stabilizer Epothilone B strongly suppresses the phenotypes induced by microtubule depolymerizing agents in human HSCs. Furthermore, chromatin immunoprecipitation assay reveals an increased binding of nuclear tubulin and TCF12 transcription factor at the CXCR4 promoter region. Depletion of TCF12 significantly suppresses microtubule depolymerization mediated upregulation of CXCR4 surface expression. These results demonstrate a previously unknown function of nuclear tubulin in regulating gene transcription through TCF12. New strategy targeting nuclear tubulin-TCF12-CXCR4 axis may be applicable to enhance HSC transplantation.

2-Arachidonoylglycerol Modulates CXCL12-Mediated Chemotaxis in Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia

To survive chemotherapy, lymphoma cells can relocate to protective niches where they receive support from the non-malignant cells. The biolipid 2-arachidonoylglycerol (2-AG), an agonist for the cannabinoid receptors CB1 and CB2, is released by stromal cells in the bone marrow. To investigate the role of 2-AG in lymphoma, we analyzed the chemotactic response of primary B-cell lymphoma cells enriched from peripheral blood of twenty-two chronic lymphocytic leukemia (CLL) and five mantle cell lymphoma (MCL) patients towards 2-AG alone and/or to the chemokine CXCL12. The expression of cannabinoid receptors was quantified using qPCR and the protein levels visualized by immunofluorescence and Western blot. Surface expression of CXCR4, the main cognate receptor to CXCL12, was analyzed by flow cytometry. Phosphorylation of key downstream signaling pathways activated by 2-AG and CXCL12 were measured by Western blot in three MCL cell lines and two primary CLL samples. We report that 2-AG induces chemotaxis in 80% of the primary samples, as well as 2/3 MCL cell lines. 2-AG induced in a dose-dependent manner, the migration of JeKo-1 cell line via CB1 and CB2. 2-AG affected the CXCL12-mediated chemotaxis without impacting the expression or internalization of CXCR4. We further show that 2-AG modulated p38 and p44/42 MAPK activation. Our results suggest that 2-AG has a previously unrecognized role in the mobilization of lymphoma cells by effecting the CXCL12-induced migration and the CXCR4 signaling pathways, however, with different effects in MCL compared to CLL.

Mycobacterium indicus pranii therapy suppresses systemic dissemination of tumor cells in B16F10 murine model of melanoma

Cancer associated morbidity is mostly attributed to the dissemination of tumor cells from their primary niche into the circulation known as “metastasis”. Mycobacterium indicus pranii (MIP) an approved immunotherapeutic agent against lung cancer (NSCLC) has shown potent anti-tumor activity in prior studies. While evaluating anti-tumor activity of MIP in mouse model, MIP treated animals typically exhibited less metastatic lesions in their pulmonary compartment. To study the role of MIP in metastasis closely, B16F10 melanoma cells were implanted subcutaneously in the mice, and the dissemination of tumor cells from the solid tumor was evaluated over a period of time. When B16F10 melanoma cells were treated with MIP in vitro, downregulation of epithelial mesenchymal transition markers was observed in these cells, which in turn suppressed the invasion, migration and adhesion of tumor cells. Notably, MIP therapy was found to be effectively reducing the metastatic burden in murine model of melanoma. Molecular characterization of MIP treated tumor cells substantiated that MIP upregulates the PPARγ expression within the tumor cells, which attenuates the NFκB/p65 levels within the nucleus, resulting in the suppression of Mmp9 expression in tumor cells. Besides that, MIP also downregulated the surface expression of chemokine receptor CXCR4 in murine melanoma cells, where chromatin immunoprecipitation confirmed the impeded recruitment of p50 and c-Rel factors to the Cxcr4 promoter, resulting in its downregulation transcriptionally. Taken together, MIP suppressed the dissemination of tumor cells in vivo, by regulating the expression of MMP9 and CXCR4 on these cells.

General anaesthetics reduce acute lymphoblastic leukaemia cell migration and homing in vitro and in vivo via CXCR4 and osteopontin mediated mechanisms

Background: Acute lymphoblastic leukaemia (ALL) is the most common type of cancer in children. General anaesthetics are often used on patients undergoing painful procedures during ALL treatments but their effects on ALL malignancy remain unknown. Herein, we aim to study the effect of two commonly used general anaesthetics, intravenous propofol and inhalational sevoflurane, on the migration and homing of ALL cells in vitro and in vivo. Methods: NALM-6 cells were treated with propofol (5 and 10 μg/ml) or sevoflurane (3.6%) in vitro for six hours. Then, cells were harvested for flow cytometry analysis. For in vitro migration experiments, NALM-6 cells were pre-treated with propofol and sevoflurane for six hours before being loaded onto the upper chamber of a migration chamber and cells were collected in the lower chamber after six hours of migration. For in vivo adhesion assays, NALM-6 cells were pre-treated with propofol and sevoflurane before an adhesion assay was carried out. In in vitro experiments, GFP-NALM-6 cells were pre-treated with propofol (10 μg/ml) or sevoflurane (3.6%) for six hours. Then, cells were injected intravenously to C57BL/6 female mice followed by intravital microscopy. Results: Both anaesthetics reduced in vitro migration, in vivo migration and in vivo homing as exemplified by 1) the reduction in the number of cells entering the bone marrow and 2) the disturbance in homing location in relation to the nearest endosteal surface. Our results indicated that general anaesthetics reduced the surface CXCR4 expression. In addition, the adhesion of leukaemia cells to thrombin cleaved osteopontin (OPN) was reduced by general anaesthetics. Those changes might result in the alterations in migration and homing. Conclusion: Together, our data suggest that both propofol and sevoflurane could reduce ALL migration and homing in vivo and in vitro via CXCR4 and OPN mediated mechanisms.

CXCR4 (CD184) Expression in Pediatric AML Is Associated with Bone Marrow Retention, Specific Disease Characteristics, and Worse Outcomes: A Report of 1004 Patients from the Children's Oncology Group AAML1031 Protocol

Introduction: CXCR4 (CD184) is a G protein-coupled chemokine receptor specific for CXCL12 that is associated with leukocyte chemotaxis. It is expressed on normal hematopoietic cells as well as hematopoietic and epithelial cancer cells. CXCR4 and CXCL12 have been associated with bone marrow retention of AML and ALL blasts and blocking this interaction via small molecule inhibitors has been investigated as a therapeutic target. CXCR4 has also been implicated in other aspects of tumor progression including metastasis, angiogenesis, and survival. As part of Children's Oncology Group protocol AAML1031 for de novo pediatric AML we prospectively evaluated CXCR4 expression by difference from normal (ΔN) flow cytometry and correlated expression with clinical characteristics and outcome. Given the impact of the bone marrow niche on the administration of chemotherapy we hypothesized that high levels of CXCR4 expression would be associated with unfavorable outcomes. Methods: In the AAML1031 phase 3 trial pediatric patients with de novo AML were randomized to receive standard chemotherapy (Arm A) or standard chemotherapy with bortezomib (Arm B), excepting patients with high allelic ratio FLT3-ITD who received standard chemotherapy plus sorafenib (Arm C). Patients were stratified as low-risk (LR) or high-risk (HR) based on cytogenetic and molecular abnormalities, and end of induction (EOI) minimal residual disease (MRD) by flow cytometry. LR patients received four cycles of the standard chemotherapy backbone while HR patients received three followed by best hematopoietic stem cell transplant. Patients who: (1) submitted a bone marrow aspirate for ΔN at diagnosis and (2) provided consent for correlative biology studies were included in this analysis. All diagnostic specimens were centrally and prospectively evaluated for the expression of CXCR4 by ΔN. Results: Surface CXCR4 expression was available for 1004 patients and expression on AML cells varied across the cohort (range: 54-17,793 molecules/cell). For analysis, the study population was divided into quartiles (n=257-258 patients each) based on expression levels. Quartiles showed no association with sex and were distributed as expected between treatment arms. Correlation with clinical characteristics showed that patients in the top quartile of CXCR4 expression were younger (5.9 vs. 11.4 years, P<0.001) and had higher bone marrow (BM) blast counts but lower peripheral blood (PB) blast counts (76% vs. 66%, p<0.001 and 33% vs. 43%, p=0.01, respectively). These patients were highly enriched for HR KMT2A fusions (43% of total fusion positive patients), as well as t(9;11) and NPM1 mutations; while they had a lower frequency of t(8;21), inv(16), and CEBPA mutations (p<0.002 for all). There was no association between CXCR4 expression and protocol risk group assignment or complete remission rates, but patients in the high expressing CXCR4 quartile were more commonly MRD negative at EOI (p=0.03). Flow cytometry also revealed a striking increase in RAM phenotype (p<0.001), with 76% of all RAM patients allocated to the CXCR4 high expressing quartile. Patients in the top quartile of CXCR4 expression had lower 5-year OS (58% vs. 66%, p=0.036) and EFS (38% vs. 47%, p=0.009) compared to other patients. CXCR4 was strongly associated with increased 5-year relapse risk (RR) (54% vs. 39%, p<0.001). Among patients with HR KMT2A fusions high expression of CXCR4 was associated with an increased 5-year RR (85% vs. 57%, p=0.04). Multivariable analysis for 5-year RR including MRD status and prognostic genetic factors indicates CXCR4 expression is independently associated with relapse, with a hazard ratio of 1.33 (95% CI 1.04-1.7; p=0.026). Conclusions: These data demonstrate that CXCR4 expression is associated with individual HR markers (RAM phenotype and HR KMT2A fusions), but not risk status across the entire study. Despite similar risk status, patients in the top quartile of CXCR4 expression had inferior outcomes. These patients had higher BM blast counts but lower PB blast counts, which has been observed in RAM phenotype, likely due to the impact of CXCR4 on bone marrow retention of leukemia cells. This increased retention may also play a role in treatment resistance, suggesting that the addition of small molecule inhibitors of CXCR4/CXCL12 to chemotherapy may help these high expressing patients, especially in subtypes such as HR KMT2A and RAM phenotype patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PIM3 kinase promotes tumor metastasis in hepatoblastoma by upregulating cell surface expression of chemokine receptor cxcr4.

Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1

CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

Inhibition of Pim Kinase Supports the Neural Stem Cell Phenotype in Neuroblastoma

INTRODUCTION: Relapse rates for high-risk neuroblastoma exceed 50%. Expression of the serine-threonine protein kinase, PIM, is associated with cancer progression and is negatively correlated with neural stemness markers in neural progenitor cells. Because promoting cellular differentiation is a component of neuroblastoma therapy, and PIM kinase negatively affects neural stemness, we aimed to determine whether PIM affected the neural stem cell phenotype in neuroblastoma. METHODS: We queried the Kocak (R2) gene database to compare PIM1 and the neural stemness marker, Nanog, in neuroblastoma. For in vitro studies, SK-N-BE(2), human neuroblastoma cells and human neuroblastoma patient–derived xenograft (PDX06) cells were treated with siRNA to achieve PIM1 kinase knockdown or with AZD1208 for PIM inhibition. Abundance of mRNA for the neural stemness marker, Nanog, was detected with quantitative reverse transcription polymerase chain reaction, and protein expression was confirmed with immunoblotting. Flow cytometry detected cell surface expression of CD133 and CXCR4, 2 additional neural stemness markers. Data were reported as mean ± SEM, with p ≤ 0.05 considered significant. RESULTS: R2 database revealed a negative correlation between PIM1 and Nanog (Fig. 1A). Protein expression of Nanog was increased following siRNA knockdown of PIM1 (Fig. 1B). AZD1208 increased the abundance of Nanog mRNA and resultant protein expression (Fig. 1C). AZD1208 treatment significantly increased cell surface expression of CD133 and CXCR4 (Fig. 1D).Figure 1CONCLUSION: Neuroblastoma cells treated with PIM inhibition dedifferentiated to a more neuronal stem cell phenotype as demonstrated by increased expression of neuronal stemness markers. These findings support further studies exploring the clinical implications of PIM kinase inhibition as an adjunct to neuroblastoma therapy.

CXCR4 mediates leukemic cell migration and survival in the testicular microenvironment.

The testis is the second most frequent extramedullary site of relapse in pediatric acute lymphoblastic leukemia (ALL). The mechanism for B-cell (B) ALL cell migration towards and survival within the testis remains elusive. Here, we identified CXCL12-CXCR4 as the leading signaling axis for B-ALL cell migration and survival in the testicular leukemic niche. We combined analysis of primary human ALL with a novel patient-derived xenograft (PDX)-ALL mouse model with testicular involvement. Prerequisites for leukemic cell infiltration in the testis were prepubertal age of the recipient mice, high surface expression of CXCR4 on PDX-ALL cells, and CXCL12 secretion from the testicular stroma. Analysis of primary pediatric patient samples revealed that CXCR4 was the only chemokine receptor being robustly expressed on B-ALL cells both at the time of diagnosis and relapse. In affected patient testes, leukemic cells localized within the interstitial space in close proximity to testicular macrophages. Mouse macrophages isolated from affected testes, in the PDX model, revealed a macrophage polarization towards a M2-like phenotype in the presence of ALL cells. Therapeutically, blockade of CXCR4-mediated functions using an anti-CXCR4 antibody treatment completely abolished testicular infiltration of PDX-ALL cells and strongly impaired the overall development of leukemia. Collectively, we identified a prepubertal condition together with high CXCR4 expression as factors affecting the leukemia permissive testicular microenvironment. We propose CXCR4 as a promising target for therapeutic prevention of testicular relapses in childhood B-ALL. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.