CXCR4 Signaling Research Articles

Abstract P5-02-25: Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition

Abstract Background: De novo resistance defined as progression within six months and acquired resistance are one of the major problems in the subset of metastatic (M), hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) patients without visceral crisis receiving CDK4/6 inhibitors (CDK4/6i) plus endocrine therapy (TX). Here, we aim to identify predictive and monitoring markers of CDK4/6i resistance by conducting transcriptional profiling of circulating tumor cells (CTCs) that represent a real-time snapshot of the heterogeneity. Methods: Blood of (A) 60 HR+/HER2- MBC patients drawn at baseline of Palbociclib plus endocrine TX (TX as first line n=31, second or more lines n=29), (B) 19 HR+/HER2- MBC patients drawn before the initiation of endocrine monoTX (control) and matched blood samples of these patients after six months under TX (n=72) and at the time of progression (n=42) were analyzed. To enlarge the global CDK4/6i cohort at baseline, blood of (C) 32 patients before the initiation of Ribociclib plus endocrine TX was also drawn. Patients with progression within six months were defined as non-responders. Isolation of CTCs was conducted using positive immunomagnetic selection (AdnaTest EMT2/StemCell Select) and preamplified cDNA was analyzed by a multimarker qPCR panel utilizing QuantiNova LNA Probe assays targeting 25 genes involved in the DNA damage -, MAPK -, STAT -, Hippo – pathway or cell cycle, chemokine sensing, multidrug resistance and cell adhesion. qPCR data was normalized to CD45 and data of 20 healthy female donor controls to identify BC CTC specific overexpression signals with a specificity of >90% for all targets. Consumables: QIAGEN, Germany. Statistical analysis included log-rank testing and univariate Cox regression. Results: For first line CDK4/6i treated patients at baseline, CETN2 and E2F1 signals correlated significantly with worse progression-free survival (PFS) while CETN2 signals also related significantly to non-response. Furthermore, CETN2 and PCNA signals were significantly associated with worse overall survival (OS). Analyzing the Palbociclib cohort after six months of TX, PCNA signals correlated significantly with a decreased PFS while EpCAM signals showed a significant association with OS. In addition, CETN2, CXCR4, EpCAM, MLH3, WWTR1 signals after six months were shown to correlate significantly with a decreased OS and PFS and MAPK1 signals were only found in the non-responders. While non-response was related to appearing (from baseline to six months under TX) ABCC2, JUN and MAPK1 signals, disappearing ABCC2 signals were only found in the responders. Dynamics of ABCC2, CXCR4, EpCAM, JUN, MAPK1, MLH3, STAT1 and WWTR1 signals from baseline to six months under TX correlated significantly with OS and CXCR4 signal dynamics significantly with a worse PFS. At the time of progression, the presence of E2F1, JUN, MAPK1 and STAT1 signals correlated significantly with a decreased OS and in comparison to baseline analysis, the prevalence of ABCC2, EpCAM, E2F1, CETN2 and CXCR4 signals increased. Conclusion: CTC overexpression signals at baseline of targets involved in the cell cycle (CETN2 and E2F1) might be predictive markers for de novo resistance to CDK4/6i as first line TX, while ABCC2 (multidrug resistance), EpCAM (cell adhesion), E2F1, CETN2 and CXCR4 (chemokine sensing) signals could indicate acquired resistance to Palbociclib. In case of disease progression, E2F1, JUN (cell cycle) and targets of the MAPK- and STAT- pathway could be relevant targets for therapeutic strategies beyond Palbociclib TX. Monitoring and prognostic value was shown for single and repeated measurement of signals under TX in genes involved in resistance, cell cycle progression, DNA damage response (MLH3, PCNA), chemokine sensing, cell adhesion and the MAPK-, STAT- and Hippo (WWTR1) pathway. Citation Format: Sabine Kasimir-Bauer, Charlotte Gruber, Stefanos Moukas, Mitra Tewes, Hans-Christian Kolberg, Rainer Kimmig, Corinna Keup. Transcriptional profiling of CTCs in metastatic breast cancer patients in the course of CDK4/6 inhibition [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-25.

A meta-analysis of chemokines in vitiligo: Recruiting immune cells towards melanocytes.

Chemokine research offers insightful information on the pathogenesis of cutaneous immune disorders, such as vitiligo. Compared to cytokines, the higher detectable levels of chemokines display promising potential as future disease biomarkers. Nonetheless, some published study results are contradictory, which can be attributed to patient characteristics and methodological differences. In this study, a meta-analysis was performed to compare chemokine expression in blood and skin samples from vitiligo patients versus healthy controls. Furthermore, the relationship between chemokine expression and disease activity was evaluated. Chemokine levels were investigated in 15 articles in the circulation and in 9 articles in vitiligo skin. Overall, some clear trends were observed. CXCR3 signaling by CXCL10 and CXCL9 has been confirmed by several reports, although CXCL10 showed more robust findings in blood samples. In this meta-analysis, CCL5, CXCL8, CXCL12, and CXCL16 levels were also significantly elevated. This indicates a complex immune pathway activation in vitiligo that overall supports a Th1-dominant response. Chemokines linked to the Th2 and Th17 pathways were less prevalent. Despite these findings, study protocols that examine a broader range of chemokines are encouraged, because current research is mostly focused on a small number of chemokines that were differentially expressed in previous studies.

Exploration of Pyrido[3,4-d]pyrimidines as Antagonists of the Human Chemokine Receptor CXCR2.

Upregulated CXCR2 signalling is found in numerous inflammatory, autoimmune and neurodegenerative diseases, as well as in cancer. Consequently, CXCR2 antagonism is a promising therapeutic strategy for treatment of these disorders. We previously identified, via scaffold hopping, a pyrido[3,4-d]pyrimidine analogue as a promising CXCR2 antagonist with an IC50 value of 0.11 µM in a kinetic fluorescence-based calcium mobilization assay. This study aims at exploring the structure-activity relationship (SAR) and improving the CXCR2 antagonistic potency of this pyrido[3,4-d]pyrimidine via systematic structural modifications of the substitution pattern. Almost all new analogues completely lacked the CXCR2 antagonism, the exception being a 6-furanyl-pyrido[3,4-d]pyrimidine analogue (compound 17b) that is endowed with similar antagonistic potency as the original hit.

Structural Basis of the Binding Mode of the Antineoplastic Compound Motixafortide (BL-8040) in the CXCR4 Chemokine Receptor

Modulation of the CXCL12-CXCR4 signaling axis is of the utmost importance due to its central involvement in several pathological disorders, including inflammatory diseases and cancer. Among the different currently available drugs that inhibit CXCR4 activation, motixafortide-a best-in-class antagonist of this GPCR receptor-has exhibited promising results in preclinical studies of pancreatic, breast, and lung cancers. However, detailed information on the interaction mechanism of motixafortide is still lacking. Here, we characterize the motixafortide/CXCR4 and CXCL12/CXCR4 protein complexes by using computational techniques including unbiased all-atom molecular dynamics simulations. Our microsecond-long simulations of the protein systems indicate that the agonist triggers changes associated with active-like GPCR conformations, while the antagonist favors inactive conformations of CXCR4. Detailed ligand-protein analysis indicates the importance of motixafortide's six cationic residues, all of which established charge-charge interactions with acidic CXCR4 residues. Furthermore, two synthetic bulky chemical moieties of motixafortide work in tandem to restrict the conformations of important residues associated with CXCR4 activation. Our results not only elucidate the molecular mechanism by which motixafortide interacts with the CXCR4 receptor and stabilizes its inactive states, but also provide essential information to rationally design CXCR4 inhibitors that preserve the outstanding pharmacological features of motixafortide.

Effect of spatial transcriptomic-derived intrinsic epithelial signatures on immune cell recruitment and behaviour in the microenvironment of colorectal cancer.

251 Background: Colorectal Cancer (CRC) remains a significant healthcare problem. Interest in the role of the Tumour Microenvironment (TME) in progression and metastasis is established, yet much remains to be learned about the mechanism of immune cell recruitment to the TME of primary and metastatic tumours. The Klintrup Makinen (KM) score measures the density of immune infiltrate to the invasive edge of tumours and is associated with good prognosis in primary CRC. Neutrophil infiltration to metastases is a poor prognostic feature suggesting phenotypic differences in the TME at primary and metastatic sites. We set out to examine epithelial determination of the TME in primary CRC. Methods: 180 patient’s primary CRC from a curated TMA underwent deep phenotyping using a multiplex-immunofluorescent panel (DAPI, Panck, CD3 (T cells), FOXP3 (T regulatory cells), CD68 (Macrophages), CD163 (Tumour associated macrophage), CD66b (Neutrophils), KI67). The Nanostring GeoMx Spatial Transcriptomic platform was used to obtain segmented (Tumour: Panck+; TME: Panck-) whole transcriptomic (18000 genes) readout from 54 matched cores from the same TMA. Results: No significant association was demonstrated between immune cell density of any cell types and clinicopathological variables including tumour side, MMR status, N-stage or survival. High KM grade was associated with high CD3 (P = 0.01) and FOXP3 (P = 0.002). Density of all immune cell types were highly corelated. Spatial transcriptomic analysis of the segmented epithelium of patients with high KM grade demonstrated over-expression of known neutrophil chemo-attractant CXCL5 (log2FC 3.76 Adj.P < 0.005). The TME compartments of cores with high CD68 and low CD163 counts were associated with adaptive immune co-ordination with high expression of CXCL13 (log2FC -2.97, Adj.P < 0.005) and CD74 (log2FC -1.42 Adj.P 0.007). IGF2 (log2FC 6.08 Adj.P < 0.005) and LCN15 (log2FC 4.96 Adj.P < 0.005) were globally over-expressed in both TME and epithelium of cores with generalised low immune infiltration. The epithelial compartment neutrophil dense cores also over-expressed IGF2 (log2FC -3.53 Adj.P 0.02). Conclusions: TMAs provide a unique opportunity for broad transcriptomic phenotyping of large numbers of patients simultaneously. The Nanostring GeoMx platform is a flexible tool which can be used in novel ways with other proteomic technologies to drive deeper analysis of the TME. CXCL5 expression in the epithelial compartment drives global immune infiltration to the TME and may be manipulated in different contexts therapeutically through CXCR2 signalling in future.

A phase I/II study of the CXCR2 inhibitor, AZD5069, in combination with durvalumab, in patients (pts) with advanced hepatocellular carcinoma (HCC).

TPS631 Background: HCC is increasing rapidly in incidence worldwide driven by a rise in chronic liver disease including non-alcoholic steato-hepatitis (NASH). Most pts are not suitable for curative or loco-regional treatments and may be candidates for systemic therapies. Immune checkpoint inhibitors combined with VEGF inhibition is a standard of care in HCC. However, a meta-analysis of 3 phase III randomised trials of PD-1 or PDL-1 inhibitors (n > 1,600 pts) with HCC suggests that pts with NASH-related HCC treated with PD-1/PDL-1 inhibitors had reduced overall survival compared with other aetiologies. Neutrophils expressing the chemokine receptor CXCR2, crucial to neutrophil recruitment in acute-injury, are highly represented in NASH-HCC. In NASH-HCC murine models, lacking response to immune-checkpoint inhibitors, AZD5069 (CXCR2 inhibitor) in combination with anti-PDL-1 suppressed tumor burden and extended survival, accompanied by an increase in tumor-associated neutrophils which switched from a pro-tumor to anti-tumor progenitor-like neutrophil phenotype. We propose that inhibition of CXCR2 may potentiate the efficacy of anti-PDL-1 inhibition in pts with HCC. Methods: In this multi-centre (n = 10) study, pts with biopsy-confirmed HCC, PS ECOG < 1, Child-Pugh A, < 1 prior systemic therapies, receive 1 of escalating doses of AZD5069 (bid, po daily) with Durvalumab (1.5 gm iv on day 1) in 28-day cycles for up to 2 years to determine the recommended phase II dose using a Keyboard design, followed by an additional cohort of pts to determine the anti-tumor efficacy of this combination using a Simon’s two-stage design (min 18, max 35 pts; target objective response rate > 30%; unacceptable response rate < 10%). Dose limiting toxicities (DLTs) are assessed during cycle 1. Disease assessments are performed 8-weekly (12-weekly after 1 year). The 1st dose cohort has been completed with no DLTs. The 2nd dose cohort opened to recruitment in September 2022. Exploratory studies (blood; pre- & on-treatment tumor and non-malignant liver biopsies) include biomarkers of CXCR2 inhibition (blood); proof-of-mechanism (tumor: expression of CXCR2, PD-L1, PD-1, CD8, CD4, CD66b, CD69); proof-of-mechanism (blood: ctDNA); drug-induced changes of mRNA expression, CXCR2 ligands & signalling pathway genes, T-cell and myeloid cell pathways, neutrophil-associated genes; predictive biomarkers (blood and tumour) include biomarkers of the CXCR2/PD-L1 immune axis; aberrant CXCR2 signalling pathways; proliferation biomarkers and CD10 (neutrophils), CD68 (macrophages), CD103 (T-regs); tumour mutational status. This study is funded by a grant from Cancer Research UK (A29287) and is co-sponsored by University of Glasgow and NHS Greater Glasgow & Clyde. Study sites are supported by the Experimental Cancer Medicine Centre Network. AZD5069 and Durvalumab are provided by Astra Zeneca. Clinical trial information: 2020-003346-36 .

The SDF-1/CXCR4 Signaling Pathway Directs the Migration of Systemically Transplanted Bone Marrow Mesenchymal Stem Cells Towards the Lesion Site in a Rat Model of Spinal Cord Injury.

It has been observed that bone marrow-derived mesenchymal stem cells (MSCs) migrate towards the injured spinal cord and promote functional recovery when systemically transplanted into the traumatized spinal cord. However, the mechanisms underlying their migration to the spinal cord remain poorly understood. In this study, we systemically transplanted GFP- and luciferase-expressing MSCs into rat models of spinal cord injury and examined the role of the stromal cell-derived factor 1 (SDF-1)/CXCR4 axis in regulating the migration of transplanted MSCs to the spinal cord. After intravenous injection, MSCs migrated to the injured spinal cord where the expression of SDF-1 was increased. Spinal cord recruitment of MSCs was blocked by pre-incubation with an inhibitor of CXCR4. Their presence correlated with morphological and functional recovery. In vitro, SDF-1 or cerebrospinal fluid (CSF) collected from SCI rats promoted a dose-dependent migration of MSCs in culture, which was blocked by an inhibitor of CXCR4 or SDF-1 antibody. The study suggests that SDF-1/CXCR4 interactions recruit exogenous MSCs to injured spinal cord tissues and may enhance neural regeneration. Modulation of the homing capacity may be instrumental in harnessing the therapeutic potential of MSCs.

CXCR4 as possible druggable target linking inflammatory bowel disease and Parkinson's disease.

Parkinson's disease (PD) is a chronic, progressive, and second most prevalent neurological disorder affecting the motor system. It has been found that people suffering with inflammatory bowel disease (IBD) are at 22% more risk for PD. In the current study, we have established a molecular link between gut and brain. The microarray gene expression datasets of Homo sapiens were obtained from Gene Expression Omnibus Database. Major genes involved in gut-brain connection were found to be CXCR4, LRRK2, APOE, SNCA, IL6, HIF-1α, ABCA1 etc. The common biological pathways linking both the pathologies were found to be HIF-signaling, cytokines interactions, JAK-STAT pathway, cholesterol metabolism, apoptosis and CXCR4 signaling which modulates the synaptic function and neuronal survival in the mature brain. It is known that flavonoid-rich foods throughout life hold the potential to limit the inflammation, neurodegeneration and, to prevent the age-dependent cognitive impairment. Therefore, the potential receptor, CXCR4 was used further for docking with twenty-seven phytochemicals from 5 different classes of Flavonoids found in several dietary items. Docking studies of the top scoring compounds were compared with a known inhibitor (BPRCX807) of receptor CXCR4 (IC50 = 40.4 ± 8.0nM). The study indicates that Flavan-3-ol families of flavonoids are the best fit and finest dietary supplements for improving brain health. Hence the food items like Pistachio nuts, hazelnuts, Green Tea, walnuts, etc. should be incorporated more in the diet of healthy people as well as in IBD and PD patients to prevent inflammation in gut and brain damage from oxidative stress.

Abstract B035: CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas

Abstract Pancreatic cancer is a fatal disease and is one of the most aggressive and metastatic malignancies worldwide. The dissemination of tumor cells is the prerequisite of metastases and correlates with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumor progression. Migrating cancer stem cells (miCSCs) characterized by CD133+ and CXCR4+ expression play a pivotal role in malignant tumor formation, have been reported to form the invasive front of the metastasis and in silico analysis showed that both CSCs and miCSCs are significantly upregulated in PDAC. However, the regulatory pattern maintaining these CSCs and especially miCSCs in PDAC remains widely elusive. Thus, this study further helps to unravel pathways responsible for maintenance of CSC (CD133+) and miCSC (CD133+ CXCR4+) population. To identify key signaling pathways responsible for both CSCs and miCSCs maintenance, we first generated a protein-protein interaction network using STRING database. Afterwards, we validated these signaling pathway(s) involved in aiding CSC and miCSC population by performing shRNA mediated knockdown of key signaling proteins in different patient-derived pancreatic cancer cell lines. Moreover, we interrogated the involvement of the tumor-stroma crosstalk in the regulation of these pathways by co-culturing tumor cells with pancreatic stellate cells. Protein-protein interaction network incorporating relevant factors involved in EMT, stemness, as well as SHH, NF-kB and AKT signaling pathway identified a strong link between the CXCL12/CXCR4 signaling axis and BMI1. Migration assay, sphere formation assay and western blot upon shRNA mediated knockdown of either CXCR4 and/or BMI1 ascertained BMI1 as a key player downstream of the CXCL12/CXCR4 axis to mechanistically effect both EMT and stemness. Pathway focused gene expression analysis as well as ELISA and immunofluorescence for CXCL12 and actin filaments, respectively, revealed the indispensable nature of tumor-stroma crosstalk on promoting CSC and miCSC population through the chemokine CXCL12. In addition, co-culture systems revealed that particularly pancreatic stellate cells play a significant role in maintaining both CSCs and miCSCs population as well as their characteristic phenotype including but not limited to chemotherapy resistance. Taken together, our results obtained in this study established mechanistically that the CXCL12/CXCR4 signaling pathway driven by tumor-stroma crosstalk not only enhances but also maintains both CSCs and miCSCs in pancreatic ductal adenocarcinomas through BMI1 ultimately promoting metastases and therapeutic resistance. Citation Format: Kanishka Tiwary, Anton Lahusen, Syeda Inaas, Stefanie Hauff, Karolin Walter, Alexander Kleger, Thomas Seufferlein, Bruno Sainz Jr., Patrick Christian Hermann. CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B035.

Cancer-associated fibroblasts contribute to cancer metastasis and apoptosis resistance in human ovarian cancer via paracrine SDF-1α.

Cancer-associated fibroblasts (CAFs), one of the main members of stromal cells in tumor microenvironment are proposed to play a central role in promoting tumor metastasis. It is unclear whether and how CAFs mediates tumor metastasis or chemoresistance in human ovarian cancer. CAFs were extracted from human ovarian cancer tissues (OCs) of patients with different kinds of histological types. We found that CAFs showed more aggressive potency than those tumor cells, both of which were isolated from the same ovarian cancer specimen. Moreover, when co-cultured with CAFs, cell migration abilities of ovarian cancer cells (SKOV3, OVCAR3 and HEY) were significantly increased. Next, we preliminarily detected a higher CAFs density in sections of metastatic lesions than those in primary tumor site of primary OCs clinically. However, no significant difference of stromal derived factors-1α (SDF-1α) production from CAFs was found between primary and metastatic lesions. Additionally, in contrast with tumor cells, CAFs exhibited obvious apoptosis resistance when treated with cisplatin. Furthermore, we found that cisplatin-induced cytotoxicity and apoptosis were significantly inhibited by co-cultured with recombinant human SDF-1α in SKOV3 in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. CAFs might be involved in the malignant metastasis in human ovarian cancer through promoting cell migration in tumor cells. And their resistance to cytotoxic agents might be mediated by paracrine SDF-1α/CXCR4 signaling in ovarian cancer.

CXCR4 signaling regulates repair Schwann cell infiltration into the spinal cord after spinal cord injury in mice

Schwann cells are glial cells that myelinate neuronal axons in the peripheral nervous system (PNS). When the PNS is damaged, Schwann cells de-differentiate into p75-positive “repair Schwann cells,” which contribute to neural circuit regeneration. Interestingly, Schwann cells in the dorsal roots are known to be reprogrammed to repair Schwann cells even after spinal cord injury (SCI) and then migrate into the injured spinal cord. However, the molecular mechanism underlying the migration of repair Schwann cells remains unknown. Since a recent in vitro study revealed the importance of CXCR4 signaling in Schwann cell migration, we investigated whether CXCR4 signaling is involved in the PNS-to-central nervous system (CNS) migration of repair Schwann cells after SCI. We revealed that repair Schwann cells express CXCR4, and its ligand CXCL12 is upregulated in the injured spinal cord. We also found that the pharmacological inhibition of CXCR4 signaling decreased the infiltration of repair Schwann cells. Moreover, CXCR4 agonist administration effectively increased the infiltration of repair Schwann cells along with improved motor function. These findings strongly suggest the involvement of CXCR4 signaling in the PNS-to-CNS migration of repair Schwann cells after SCI.

Age-related liver endothelial zonation triggers steatohepatitis by inactivating pericentral endothelium-derived C-kit.

Aging leads to systemic metabolic disorders, including steatosis. Here we show that liver sinusoidal endothelial cell (LSEC) senescence accelerates liver sinusoid capillarization and promotes steatosis by reprogramming liver endothelial zonation and inactivating pericentral endothelium-derived C-kit, which is a type III receptor tyrosine kinase. Specifically, inhibition of endothelial C-kit triggers cellular senescence, perturbing LSEC homeostasis in male mice. During diet-induced nonalcoholic steatohepatitis (NASH) development, Kit deletion worsens hepatic steatosis and exacerbates NASH-associated fibrosis and inflammation. Mechanistically, C-kit transcriptionally inhibits chemokine (C-X-C motif) receptor (CXCR)4 via CCAAT enhancer-binding protein α (CEBPA). Blocking CXCR4 signaling abolishes LSEC-macrophage-neutrophil cross-talk and leads to the recovery of C-kit-deficient mice with NASH. Of therapeutic relevance, infusing C-kit-expressing LSECs into aged mice or mice with diet-induced NASH counteracts age-associated senescence and steatosis and improves the symptoms of diet-induced NASH by restoring metabolic homeostasis of the pericentral liver endothelium. Our work provides an alternative approach that could be useful for treating aging- and diet-induced NASH.

Tumor-associated macrophages promote resistance of hepatocellular carcinoma cells against sorafenib by activating CXCR2 signaling

BackgroundSorafenib (SOR) is the first line treatment for advanced hepatocellular carcinoma (HCC), but resistance develops frequently. Tumor-associated macrophages (TAMs) have been reported to affect the progression of HCC. We therefore aimed to study the role of TAMs in promoting SOR resistance.MethodsImmunofluorescence staining for the M2 marker CD204 and the cancer stem cell (CSC) markers CD44 and CD133 was performed in paired HCC and adjacent noncancerous tissues and HCC tissues stratified by response of SOR treatment. HCC/U937 coculture system and cytokines were used to induce M2 polarization for studying the effects of M2 TAMs on CSC properties and apoptotic death of HCC cells after SOR treatment.ResultsHigher expression of CD204, CD44, and CD133 was observed in patients with SOR nonresponse (SNR) than in those with SOR response (SR), suggesting that SNR is positively correlated to levels of CSCs and M2 TAMs. After coculture, M2 TAMs could increase the level of CSCs but decrease SOR-induced apoptosis. Incubation of HCC cells with coculture conditioned medium increased the formation of spheres that were resistant to SOR. Furthermore, CXCL1 and CXCL2 were found to be the potential paracrine factors released by M2 TAMs to upregulate SOR resistance in HCC cells. Treatment with CXCL1 and CXCL2 could increase HCC CSC activity but decrease SOR-induced apoptosis by affecting BCL-2 family gene expression. Using pharmacological inhibitors, CXCR2/ERK signaling was found to be critical to CXCL1- and CXCL2-mediated SOR resistance.ConclusionThis study identified CXCL1, CXCL2, and their downstream CXCR2/ERK signaling as potential therapeutic targets to overcome SOR resistance in HCC.

Astragaloside IV as a novel CXCR4 antagonist alleviates osteoarthritis in the knee of monosodium iodoacetate-induced rats

Background and PurposeC-X-C chemokine receptor type 4 (CXCR4) inhibition protects cartilage in osteoarthritis (OA) animal models. Therefore, CXCR4 has becoming a novel target for OA drug development. Since dietary and herbal supplements have been widely used for joint health, we hypothesized that some supplements exhibit protective effects on OA cartilage through inhibiting CXCR4 signaling. MethodsThe single-cell RNA sequencing data of OA patients (GSE152805) was re-analyzed by Scanpy 1.9.0. The docking screening of CXCR4 antagonists was conducted by Autodock Vina 1.2.0. The CXCR4 antagonistic activity was evaluated by calcium response in THP-1 cells. Signaling pathway study was conducted by bulk RNA sequencing and western blot analysis in human C28/I2 chondrocytes. The anti-OA activity was evaluated in monosodium iodoacetate (MIA)-induced rats. ResultsAstragaloside IV (ASN IV), the predominate phytochemical in Astragalus membranaceus, has been identified as a novel CXCR4 antagonist. ASN IV reduced CXCL12-induced ADAMTS4,5 overexpression in chondrocytes through blocking Akt signaling pathway. Furthermore, ASN IV administration significantly repaired the damaged cartilage and subchondral bone in MIA-induced rats. ConclusionThe blockade of CXCR4 signaling by ASN IV could explain anti-OA activities of Astragalus membranaceus by protection of cartilage degradation in OA patients. Since ASN IV as an antiviral has been approved by China National Medical Products Administration for testing in people, repurposing of ASN IV as a joint protective agent might be a promising strategy for OA drug development.

CXCR4 Promotes Bortezomib Resistance and Regulates Metabolic Reprogramming, Activating Both Glycolytic and Oxphos Pathways in Multiple Myeloma

CXCR4 overexpression in multiple myeloma (MM) correlates with aggressive features and poor prognosis. Our previous work has demonstrated that CXCR4 induces proliferation and protect MM cells from drug-induced apoptosis. In the current study we addressed the role of CXCR4-mediated signals in MM cell metabolism and drug response, using MM cell lines with low CXCR4 versus exogenously overexpressed CXCR4, and cells with acquired resistance to bortezomib (Bort) established in our laboratory. MM cells with overexpressed CXCR4 exhibited a higher expression of numerous components of the CXCR4 signaling, including pro-survival MEK and PI3K/AKT/mTOR pathways, with constitutively phosphorylated AKT, pS6, and 4E-BP1 proteins. Given the role of mTOR in glycolysis regulation, elevated levels of key enzymes including HK2, LDHA1, and PDK1 were detected in cells with up-regulated CXCR4, indicating increased glycolytic activity. Importantly, CXCR4-overexpressing MM cells demonstrated reduced responsiveness to 2-DG and DCA, known inhibitors of glycolysis. In contrast to Bort-sensitive cells, CXCR4-expressing resistant cells demonstrated only transient growth inhibition rather than apoptosis in response to inhibition of glycolysis with less mitochondrial depolarization, and growth recovery, once the glycolysis inhibitors were removed. These results suggest that MM cells with high CXCR4 expression and those resistant to Bort, are less dependent on glycolysis and may activate alternative metabolic pathways that support cell adaptation and survival. Importantly, increased mitochondrial mass, enhanced mitochondrial membrane potential, and augmented ATP production were found in both CXCR4-high and Bort-resistant MM cells, suggesting that CXCR4 can induce mitochondrial fitness and associated metabolic pathways including oxidative phosphorylation (OXPHOS) in MM. In addition to energy production, OXPHOS coupled with electron transport chain (ETC) generates reactive oxygen species (ROS). Indeed, MM cells with high CXCR4 possessed elevated basal ROS levels. Notably, Bort treatment induced distinct molecular responses in CXCR4-low sensitive versus CXCR4-high Bort-resistant cells. In sensitive cells, Bort reduced AKT/mTOR activity with a concomitant decrease in mRNA levels of HK2 and LDHA, suggesting Bort to mediate an anti-glycolytic effect. In contrast, expression levels of glycolytic genes were not affected by Bort in resistant cells. Furthermore, Bort reduced ATP levels and mitochondrial polarization in sensitive cells, while increasing both in CXCR4-high resistant MM cells, therefore indicating a metabolic switch to OXPHOS in resistant cells upon Bort exposure. Analysis of molecular function profile revealed a distinct proteomic signature in purified mitochondria from MM cells with overexpressed CXCR4, and cells with acquired resistance to Bort, associated with enhanced expression of proteins involved in the metabolic processes and antioxidant activity. Thus, abundant proteins involved in respiratory activity were upregulated in CXCR4-high resistant cells, including ETC Complex II, III and IV subunits. Furthermore, an increase in mitochondrial ribosomal proteins, metabolite transporters, proteasome subunits, outer membrane proteins involved in mitochondria stabilization, ROS detoxification proteins, and factors regulating mitochondrial dynamics (both fission and fusion) was observed in Bort resistant cells with high CXCR4. Altogether, these results suggest that CXCR4-regulated chemoresistance is driven by empowered mitochondria with increased OXPHOS, stabilized membrane, and enhanced ROS detoxifying capacity, while CXCR4 low cells are vulnerable to mitochondrial oxidative stress. In approval, using a selective and potent OXPHOS inhibitor, IACS010759 monotherapy or in combination with Bort effectively targeted Bort-resistant cells, inducing overwhelming oxidative stress and uncompensated mitochondrial damage. Collectively, our findings indicate that CXCR4-mediated chemoresistance in MM cells involves AKT/mTOR signaling, promoting both glycolysis and mitochondrial OXPHOS, and enabling metabolic plasticity with OXPHOS shift upon Bort treatment. These results delineate the novel mechanisms in CXCR4 activity and suggest OXPHOS as a potential target for therapeutic intervention in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMU-43. CXCR3 SIGNALING ENGAGES GLIOMA INFILTRATING T CELLS AND IS REQUIRED FOR THE ANTITUMOR EFFICACY OF INNATE STIMULATING IMMUNOTHERAPY

Abstract BACKGROUND T cell dysfunction is well documented in patients with gliomas, yet mechanisms explaining T cell dysfunction in gliomas are lacking. Recent evidence links CXCR3 signaling and functional immune surveillance in both preclinical models and human data sets. METHODS Bulk and single cell RNA-seq data sets were used to compare CXCR3 ligand expression in gliomas to that of other solid tumor types. The role of CXCR3 signaling in mediating the recruitment and phenotype of tumor infiltrating lymphocytes (TILs) was tested using CXCR3 blockade and genetic deletion of CXCR3 in murine tumor models. The capacity of relevant innate stimulating immunotherapies to induce CXCR3 ligands in human glioma tissue was assessed using an ex vivo glioma slice culture assay, and the contribution of CXCR3 signaling to polio virotherapy (PVSRIPO) and STING agonist therapy was tested in murine models. RESULTS Relative to other solid tumors, gliomas express significantly lower levels of CXCR3 ligands (CXCL9, 10, and 11), which are largely expressed by tumor associated myeloid cells. Blocking CXCR3 signaling profoundly restricted TIL recruitment in murine gliomas, and resulted in decreased TIL stemness features coinciding with exacerbated exhaustion phenotypes. Polio virotherapy and STING agonism efficiently induced CXCR3 ligand production in fresh human glioma tissue in a manner dependent upon myeloid cells, and at levels similar to that induced by the same agents in melanoma and breast cancer tissue. CXCR3+ TILs expressed higher levels of memory and activation markers, proliferated more, and were required for the antitumor efficacy of both polio virotherapy and a STING agonist in mice. CONCLUSION This work indicates that CXCR3 signaling, a key contributor to cancer immune surveillance, is disrupted in human gliomas relative to other solid tumor types. Intratumor virotherapy and STING agonists hold potential to rectify diminished CXCR3 ligand expression in gliomas.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

CXCR4 expression is associated with proneural-to-mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor and consists of three molecular subtypes: proneural (PN), mesenchymal (MES) and classical (CL). Transition between PN to MES subtypes (PMT) is the glioma analog of the epithelial-mesenchymal transition (EMT) in carcinomas and is associated with resistance to therapy. CXCR4 signaling increases the expression of MES genes in glioma cell lines and promotes EMT in other cancers. RNA sequencing (RNAseq) data of PN GBMs in The Cancer Genome Atlas (TCGA) and secondary high-grade gliomas (HGGs) from an internal cohort were examined for correlation between CXCR4 expression and survival as well as expression of MES markers. Publicly available single-cell RNA sequencing (scRNAseq) data was analyzed for cell type specific CXCR4 expression. These results were validated in a genetic mouse model of PN GBM. Higher CXCR4 expression was associated with significantly reduced survival and increased expression of MES markers in TCGA and internal cohorts. CXCR4 was expressed in immune and tumor cells based on scRNAseq analysis. Higher CXCR4 expression within tumor cells on scRNAseq was associated with increased MES phenotype, suggesting a cell-autonomous effect. In a genetically engineered mouse model, tumors induced with CXCR4 exhibited a mesenchymal phenotype and shortened survival. These results suggest that CXCR4 signaling promotes PMT and shortens survival in GBM and highlights its inhibition as a potential therapeutic strategy.

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.

KDM6A Loss Recruits Tumor-Associated Neutrophils and Promotes Neutrophil Extracellular Trap Formation in Pancreatic Cancer.

KDM6A loss in pancreatic cancer cells alters the immune microenvironment by increasing CXCL1 secretion and neutrophil recruitment, providing a rationale for targeting the CXCL1-CXCR2 signaling axis in tumors with low KDM6A.