CXCR4 Inhibitor Research Articles

Exosomal IL-8 derived from Lung Cancer and Colon Cancer cells induced adipocyte atrophy via NF-κB signaling pathway

BackgroundCytokines secreted in the tumor microenvironment function in cancer cachexia (CC), a common clinicopathological syndrome associated with adipocyte wasting and skeletal muscle atrophy. Extracellular vesicles (EVs) secreted by cancer cells actively engage in inter-tissue communication; EVs and enclosed cytokines are largely undefined in CC adipocytes wasting.MethodsEVs derived from Lewis lung carcinoma (LLC) and colorectal cancer C26 cells were extracted and characterized. Conditioned medium and EVs from cancer cells were applied to 3 T3-L1 adipocytes. Recombinant IL-8, IL-8 neutralizing antibody, CXCR2 and NF-κB inhibitor were examined in functional assays. Lipolysis of adipocytes was monitored by Western blots, Oil red O staining and glycerol assays. Furthermore, LLC and C26 cell lines were established as cachexia model to explore the relevance of IL-8 and NF-κB signaling in CC adipose wasting. Adipose tissues were collected for histology analyses.ResultsLLC and C26 cell-derived EVs induced lipolysis of 3 T3-L1 adipocytes. Specially, Dil-labeled EVs were effectively taken up by 3 T3-L1 adipocytes, which were motivated by the delivered IL-8 to elicit the NF-κB pathway. In comparison, special IL-8 neutralizing antibody relieved that lipolysis of 3 T3-L1 adipocytes induced by EVs together with conditioned medium of LLC and C26 cells, respectively. Consistently, both CXCR2 and NF-κB inhibitors would lessen the phenotype of lipolysis in 3 T3-L1 adipocytes. In the in vivo settings, both LLC and C26-tumor bearing mice had higher serum IL-8 levels as compared to the control groups. Two typical lipolysis markers, PGC1α and UCP1, were also up-regulated in the adipose tissues of LLC and C26-tumor mice groups, respectively.ConclusionsEVs secreted by LLC and C26 tumor cells would induce adipocyte wasting via extracellular IL-8-mediated NF-κB signaling. Our study pointed out the physiological and therapeutic values of exosomal IL-8 in CC lipolysis.

Involvement of CXCL17 and GPR35 in Gastric Cancer Initiation and Progression.

The expression of CXC motif chemokine 17 (CXCL17) and its reported membrane receptor G-protein-coupled receptor 35 (GPR35) in different gastric pathological lesions and their clinical implications are largely unknown. In this study, a total of 860 pathological sections were immune-stained with either anti-CXCL17 or anti-GPR35 antibodies. Their expression was scored within the area of the normal gastric gland of non-atrophic gastritis (NAG-NOR), intestinal metaplasia of atrophic gastritis (AG-IM), IM adjacent to GC (GC-IM), and GC tissue. The clinical significance and potential function of CXCL17 and GPR35 were explored using multiple methods. Our results suggested that CXCL17 expression was gradually upregulated during the pathological progress of gastric diseases (NAG-NOR < AG-IM < GC-IM), but significantly downregulated when GC occurred. GPR35 had a similar expression pattern but its expression in GC remained abundant. High CXCL17 expression in GC was associated with less malignant behavior and was an independent biomarker of favorable prognosis. Overexpressing CXCL17 in HGC27 cells significantly upregulated CCL20 expression. TCGA analysis identified that CXCL17 was negatively correlated with some cancer-promoting pathways and involved in inflammatory activities. CTRP analysis revealed that gastric cell lines expressing less CXCL17 and were more sensitive to the CXCR2 inhibitor SB-225002.

ADAM12 abrogation alters immune cell infiltration and improves response to checkpoint blockade therapy in the T11 murine model of triple-negative breast cancer

ABSTRACT Immunosuppressive tumor microenvironment (TME) impedes anti-tumor immune responses and contributes to immunotherapy resistance in triple-negative breast cancer (TNBC). ADAM12, a member of cell surface metalloproteases, is selectively upregulated in mesenchymal/claudin-low TNBCs, where its expression is largely restricted to tumor cells. The role of cancer cell-expressed ADAM12 in modulating the immune TME is not known. We show that Adam12 knockout in the T11 mouse syngeneic transplantation model of claudin-low TNBC leads to decreased numbers of tumor-infiltrating neutrophils (TINs)/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and increased numbers of tumor-infiltrating B cells and T cells. ADAM12 loss in cancer cells increases chemotaxis of B cells in vitro and this effect is eliminated by inhibition of CXCR4, a receptor for CXCL12, or anti-CXCL12 blocking antibody. Importantly, ADAM12 loss in T11 cancer cells sensitizes tumors to anti-PD1/anti-CTLA4 combination therapy, although the initial responsiveness is followed by acquired therapy resistance. Depletion of B cells in mice eliminates the improved response to immune checkpoint blockade of Adam12 knockout T11 tumors. Analysis of gene expression data for claudin-low TNBCs from the METABRIC patient cohort shows significant inverse correlations between ADAM12 and gene expression signatures of several anti-tumor immune cell populations, as well as a significant positive correlation between ADAM12 and gene expression signature of TINs/PMN-MDSCs. Collectively, these results implicate ADAM12 in immunosuppression within the TME in TNBC.

Genetics of severe congenital neutropenia as a gateway to personalized therapy

Severe congenital neutropenias (SCNs) are rare diseases, and to date about 30 subtypes have been described according to their genetic causes. Standard care aims to prevent infections and limit the risk of leukemic transformation; however, several subtypes may have additional organ dysfunction(s), requiring specialized care. Granulocyte colony-stimulating factor and hematopoietic stem cell transplantation are now the bedrock of standard care. Better understanding of SCN mechanisms now offers the possibility of adapted therapy for some entities. An inhibitor of sodium glucose cotransporter, an antidiabetic drug, may attenuate glycogen storage disease type Ib and glucose-6-phosphatase catalytic subunit 3 neutropenias by clearing 1,5-anhydroglucitol, the precursor of the phosphate ester responsible for these SCNs. Chemokine receptor CXCR4 inhibitors contribute to reversing the leukocyte defect in warts, hypoglobulinemia, infections, and myelokathexis syndrome. All these new approaches use oral drugs, which notably improve quality of life. Additionally, improved research into clonal evolution has highlighted some ways to potentially prevent leukemia, such as stimulating somatic genetic rescue, a physiological process that might limit the risk of leukemic transformation.

MiR-301a Deficiency Attenuates the Macrophage Migration and Phagocytosis through YY1/CXCR4 Pathway.

(1) Background: the miR-301a is well known involving the proliferation and migration of tumor cells. However, the role of miR-301a in the migration and phagocytosis of macrophages is still unclear. (2) Methods: sciatic nerve injury, liver injury models, as well as primary macrophage cultures were prepared from the miR-301a knockout (KO) and wild type (WT) mice to assess the macrophage's migration and phagocytosis capabilities. Targetscan database analysis, Western blotting, siRNA transfection, and CXCR4 inhibition or activation were performed to reveal miR301a's potential mechanism. (3) Results: the macrophage's migration and phagocytosis were significantly attenuated by the miR-301a KO both in vivo and in vitro. MiR-301a can target Yin-Yang 1 (YY1), and miR-301a KO resulted in YY1 up-regulation and CXCR4 (YY1's down-stream molecule) down-regulation. siYY1 increased the expression of CXCR4 and enhanced migration and phagocytosis in KO macrophages. Meanwhile, a CXCR4 inhibitor or agonist could attenuate or accelerate, respectively, the macrophage migration and phagocytosis. (4) Conclusions: current findings indicated that miR-301a plays important roles in a macrophage's capabilities of migration and phagocytosis through the YY1/CXCR4 pathway. Hence, miR-301a might be a promising therapeutic candidate for inflammatory diseases by adjusting macrophage bio-functions.

Advanced EPI-X4 Derivatives Covalently Bind Human Serum Albumin Resulting in Prolonged Plasma Stability

Advanced derivatives of the Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) have shown therapeutic efficacy upon topical administration in animal models of asthma and dermatitis. Here, we studied the plasma stability of the EPI-X4 lead compounds WSC02 and JM#21, using mass spectrometry to monitor the chemical integrity of the peptides and a functional fluorescence-based assay to determine peptide function in a CXCR4-antibody competition assay. Although mass spectrometry revealed very rapid disappearance of both peptides in human plasma within seconds, the functional assay revealed a significantly higher half-life of 9 min for EPI-X4 WSC02 and 6 min for EPI-X4 JM#21. Further analyses demonstrated that EPI-X4 WSC02 and EPI-X4 JM#21 interact with low molecular weight plasma components and serum albumin. Albumin binding is mediated by the formation of a disulfide bridge between Cys10 in the EPI-X4 peptides and Cys34 in albumin. These covalently linked albumin-peptide complexes have a higher stability in plasma as compared with the non-bound peptides and retain the ability to bind and antagonize CXCR4. Remarkably, chemically synthesized albumin-EPI-X4 conjugates coupled by non-breakable bonds have a drastically increased plasma stability of over 2 h. Thus, covalent coupling of EPI-X4 to albumin in vitro before administration or in vivo post administration may significantly increase the pharmacokinetic properties of this new class of CXCR4 antagonists.

GPC-100 Is a Novel CXCR4 Inhibitor That Leads to Improved in Vitro Potency and In Vivo Efficacy in Stem Cell Mobilization When Combined with a Beta-Blocker

GPC-100 Is a Novel CXCR4 Inhibitor That Leads to Improved in Vitro Potency and In Vivo Efficacy in Stem Cell Mobilization When Combined with a Beta-Blocker

A Phase I Safety and Feasibility Study to Evaluate Motixafortide (CXCR4/SDF-1 Inhibition) and Natalizumab (VLA-4/VCAM-1 Inhibition) As a Novel Regimen to Mobilize CD34+ Hematopoietic Stem Cells for Gene Therapy in Sickle Cell Disease

Background: Sickle cell disease (SCD) is one of the most common genetic diseases globally. Clinical manifestations of SCD commonly include anemia, pain, vaso-occlusive events and cumulative end organ damage leading to significant morbidity and mortality. Historically, allogeneic hematopoietic cell transplantation (HCT) represented the only curative therapy for SCD, but is associated with significant HCT-related morbidity and mortality which limit its use (PMID: 8663884, PMID: 20007560). Autologous hematopoietic stem cell (HSC) -based gene therapies now offer curative potential with decreased toxicity (PMID: 28249145). However, effective HSC-based gene therapy depends on collection of sufficient HSCs (ideally >10x106 CD34+ cells/kg), typically from peripheral blood (PB) (PMID: 33956057). G-CSF and CXCR4 inhibition (CXCR4i) with plerixafor are the most widely used mobilization strategies (PMID: 19363221, PMID: 19720922). However, G-CSF is associated with risk of fatal vaso-occlusive events in SCD (PMID: 9734950, PMID: 11368061, PMID: 19513902). Meanwhile, short-acting CXCR4i with plerixafor alone does not reliably yield optimal HSC numbers for gene therapy applications (PMID: 30282642, PMID: 29419425, PMID: 29472357). Therefore, developing novel HSC mobilization regimens to rapidly and reliably mobilize optimal CD34+ HSCs for gene therapy in SCD represents an unmet need. Motixafortide (M) is a high-affinity, long-acting CXCR4 inhibitor. The recent Phase III, randomized, controlled GENESIS Trial demonstrated combination M+G-CSF resulted in 88.8% of patients collecting ≥6x106 CD34+ cells/kg (median 10.8x106) within 1 leukapheresis (LP) for HCT. This compares favorably to historical data with plerixafor+G-CSF where 54.2% collected to goal in 1 LP. Motixafortide also mobilized more immunophenotypically primitive HSCs and multi-potent progenitors compared to plerixafor. Meanwhile, transcriptional profiling by single-cell RNA sequencing revealed HSCs mobilized with M have gene expression profiles associated with enhanced regeneration and self-renewal, compared to G-CSF or plerixafor (DOI: 10.1182/blood-2021-144296). Motixafortide alone also mobilized an average of 11.6×106 CD34+ cells/kg in healthy volunteers, while mobilizing 91.6% of allogeneic HSC donors to goal in ≤2 LPs (median 6.07x106) (DOI: 10.1182/blood-2018-99-109701, PMID: 28835380). Natalizumab (N) is a monoclonal antibody to the a4 integrin subunit of a4b1 (VLA-4) capable of VLA4 inhibition (VLA4i). In mice and primates, N led to an ~8-fold increase in PB CD34+ HSCs in 24 hrs (PMID: 7692447). In humans, a single N infusion resulted in a 5- to 6-fold increase PB CD34+ HSCs. Meanwhile, the average PB CD34+ HSC levels after a single dose were similar to levels with repeated doses, indicating rapid mobilization of maximal PB HSCs by 24 hrs after N infusion (PMID: 18195093). Combination CXCR4i and VLA-4i has been well-tolerated and resulted in synergistic mobilization of HSCs in preclinical studies (PMID: 21886173). Therefore, we hypothesize that combination CXCR4i and VLA-4i with M+N represents a safe and effective strategy for robust, rapid and synergistic mobilization of HSCs to PB in patients with SCD. Study Design and Methods: This is a single center, single-arm, dose de-escalation, Phase 1 safety and feasibility study. Key eligibility criteria include patients with SCD (SS or Sβ0) age 18-35 years receiving automated RBC exchanges. Patients with prior HCT or gene therapy will be excluded. We estimate 5 patients in this pilot feasibility study will provide sufficient safety and preliminary efficacy data to guide further development of this novel mobilization strategy. All patients will have baseline labs prior to M (1.25mg/kg, subcutaneous), N (300mg, IV) and M+N dosing. Each patient will then undergo mobilization and LP (1 blood volume), per protocol (see Figure 1). The primary outcome will be to assess the safety and tolerability of M, N and M+N in each SCD patient, defined by dose-limiting toxicities and maximum tolerated dose. Key secondary outcomes include: kinetics of CD34+ HSC mobilization to PB (CD34+ cells/uL), number of CD34+ HSCs collected via LP (CD34+ cells/kg/L), incidence of AEs, pharmacokinetics and the recommended phase 2 dose. Correlative studies include immunophenotypic and single cell transcriptional profiling of CD34+ HSCs from LP product mobilized with M, N and M+N. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Efficacy of Same Day Plerixafor Dosing on CD34+ Haematopoietic Stem Cell Collection: A Single Centre Australian Experience

Introduction: Haematopoietic stem cell (HSC) transplantation is an important therapeutic option for many haematological conditions, as well as, some solid organ cancers. Plerixafor (a selective inhibitor of CXCR4) can be used in patients for primary stem cell mobilisation; those who have previously failed stem cell mobilisation ("rescue" or "Planned"); or in patients with a poor peripheral CD34+ response to GCSF and/or chemotherapy mobilisation ("on demand", "just in time" or "pre-emptive"). In Australia, Plerixafor is funded for patients with CD34+ count <10 cells/µl on day of collection or if first apheresis has yielded CD34+ <1 x 106cells/kg. In these patients, Plerixafor is administered the evening prior to the planned morning collection but this presents logistical issues as it has to be given after hours. Therefore, it was proposed to administer Plerixafor followed by same day HSC collection to improve the logistics of these "just in time" collections. Methods: A retrospective case note audit identified 96 patients who received Plerixafor for HSC collection between 03/2017 and 06/2021. Patients were categorised into PM (late afternoon/evening dosing) and AM (morning dosing). Transplant HSC dose target was >2 x 106/kg. Results: Of the 96 patients, 17 received 2 doses of plerixafor with 4 of these patients receiving both an AM and PM dose. These 4 patients were excluded from the final analysis. Of the 92 patients remaining, 57% (n=52) of patients received PM and 43% (n=40) AM dosing. Median age was 64 years (range 23 to 78). 58% were male (n=53) and 42% female (n=39). Underlying malignancy included Myeloma (n=51), Lymphoma (n=37) and Solid malignancy (n=4). Median pre plerixafor blood CD34+ count was 8.7x10^6/kg (range 0.3-35.5) in the PM group and 9.2 (range 1.6-38.2) in AM group prior to collection. Median number of HSCs collected post-plerixafor administration was 4.97 x106/kg (range 0.91-34.97) for PM group vs 4.16 x106/kg (range 0.71-9.52) AM group. Total median HSC number collected for patients in the PM group was 6.6x106cells/kg (range 3.14-8.18) vs 5.16x106/kg (range = 2.46-6.45) in the AM group. The AM group had an increased median HSC collected post plerixafor of 83% vs PM median of 75%. A transplantable HSC dose was collected in 97% of those in the AM group and 96% in the PM group. Median number of apheresis procedures with PM dosing was 2 (range 1-5) vs 2.4 with AM dosing (range 1-4). Conclusion: While post plerixafor HSC collection doses were higher with PM dosing this is countered by the total median HSC collection being higher in the AM group. These findings show that same day plerixafor dosing is a feasible option with similar numbers of patients achieving their target HSC collection.

Polymeric CXCR4 Inhibitors Exhibit Novel Anti-Leukemic Effects

Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are common leukemias with a mortality rate estimated to be greater than 50%. This dismal outcome is in part due to current therapeutics. Intensive remission therapy is difficult for patients to tolerate and despite good complete remission rates, is not curative for all patients. In relapse/refractory status, there are even fewer effective therapeutics. There is a strong need for more effective, less toxic therapeutics. The surface receptor CXCR4 has become a recent target of interest, as it is highly expressed on leukemia cells and, via its canonical ligand CXCL12, enables cells to evade chemotherapeutics by localizing them to the protective bone marrow niche. The small molecule inhibitor AMD3100 (plerixafor) disrupts this localization by blocking the CXCL12:CXCR4 interaction and is being investigated for its ability to mobilize leukemia cells to the peripheral blood. Thus, AMD3100 acts as a chemosensitizer, but by itself, AMD3100 does not have anti-leukemic effects. It has been recently shown that CXCR4 has additional non-canonical activities outside of CXCL12 that are vital for leukemia survival and maintenance. Genetic knock out of Cxcr4 in an AML model decreases survival and increases differentiation in a CXCL12 independent manner. This implies that CXCR4 has previously unrecognized activities in leukemia and that inhibitors capable of blocking both CXCR4's canonical and non-canonical activities would more effective therapeutics. We developed a class of polymeric CXCR4 inhibitors (PAMDs) via Michael-type addition of AMD3100 and focused on one analog PAMD-Ch17, which contains additional cholesterol modifications. PAMD-Ch17 was originally designed to delivery siRNAs, but we surprisingly found it had anti-leukemic effects on its own. PAMD-Ch17, but not AMD3100, induces cytotoxicity and promotes differentiation of mouse AML cells in vitro. In colony assays, treatment with PAMD-Ch17 causes a statistically significant decrease in leukemia colony growth while treatment with AMD3100 was indistinguishable from the control. Across a panel of seven human AML and ALL cell lines, PAMD-Ch17 inhibited metabolic activity, a proxy for viability, at the nanomolar doses whereas AMD3100 had no effect even at the highest micromolar dose. As we predict that PAMD-Ch17 is acting through CXCR4, we compared cell surface expression of the receptor to PAMD-Ch17 sensitivity for each cell line. We found that PAMD-Ch17 IC50 was moderately negatively correlated with CXCR4 expression (r= -0.69), suggesting PAMD-CH17 mediates at least some of its effects through CXCR4. To further address the mechanisms of PAMD-Ch17's activity, we cultured Jurkat T-ALL cells with AMD3100 or PAMD-Ch17 at the same dose for 24 hours and stained using the 12G5 CXCR4 monoclonal antibody, which is known to compete with AMD3100 and CXC12. We found that AMD3100 caused a statistically significant decrease in 12G5 staining as compared to control treated cells, but PAMD-Ch17 induced a complete loss of 12G5 staining that was statistically significant as compared to both control and AMD3100 treated cells. PAMD-Ch17, but not AMD3100, also caused a moderate, but significantly decrease in staining with another antibody against the N-terminus of CXCR4, a region not predicted to be affected by AMD3100 or CXCL12. These results indicate that PAMD-Ch17 more effectively blocks the epitope recognized by the 12G5 antibody but may also interact other domains of CXCR4 or decrease its cell surface expression. We also developed a line of PAMD-CH17 resistant Jurkat cells. The resistant cells have an IC50 of at least 2-fold greater than naïve Jurkat cells. Interestingly, the resistant cells are not recognized by the 12G5 monoclonal antibody but are stained by the N-terminus antibody. These results imply that the CXCR4 expressed on resistant cells has an altered conformation that may less effectively bind PAMD-Ch17. Collectively, these data suggest that PAMD-Ch17 more effectively inhibits CXCR4 leading to its novel anti-leukemic effects and support the development of PAMDs for the treatment of leukemia.

Abstract C031: Targeting granulocytic MDSC-derived inflammasome activation to overcome stromal inflammation in pancreatic cancer

Abstract Objective: The major drivers of therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) are myeloid cell-derived signaling that sustains immune tolerance/exclusion, and interleukin-1 (IL-1)-mediated inflammatory polarization of cancer-associated fibroblasts (iCAF) which promotes stromal inflammation by elaborating soluble factors (i.e., CXCL1, IL-6) that further accelerate myeloid chemotaxis. Moreover, we have recently shown that enrichment of pathways related to inflammasome activation-which involves recruitment of ASC complexes culminating in IL-1β generation—is a major contributor to chemoresistance in advanced PDAC patients. However, how these disparate pathways converge to mediate stromal inflammation in PDAC is incompletely understood. Methods: Single-cell RNA sequencing (scRNAseq) and caspase-1 luminescence assays in human and genetically engineered mouse (GEM) PDAC models were interrogated to identify cellular source of inflammasome-derived IL-1β. Gene set enrichment analysis in bulk RNA-sequencing data and signal transduction studies examined novel pathways associated with inflammasome activation in granulocytic myeloid-derived suppressor cells (gMDSC). scRNAseq and ASC-speck formation via confocal microscopy in intratumoral gMDSCs was performed in PKT mice treated with a novel anti-ASC antibody. Results: scRNAseq in human and GEM PDAC models revealed gMDSCs as the dominant source of inflammasome activation-derived IL1B expression. Functionally, caspase-1 activation and ASC-speck formation was strongest in intratumoral gMDSCs, compared with tumor-cell, CAF, macrophage, T-cell, and dendritic cell, compartments. Investigating developmental trajectories of single-cell transcriptomes in intratumoral gMDSCs from Panc02 tumors revealed an activated Cd14+ gMDSC state with strong co-expression of Cxcr2 and Il1b. As such, treatment of PKT GEM and orthotopically injected KPC tumor-bearing mice with CXCR2 inhibitor AZD5069 significantly abrogated inflammasome activation in intratumoral and splenic gMDSCs. In vitro signal transduction and RNA-seq studies revealed cooperativity between Tlr4-Myd88 and Cxcr2-Tpl2-p38 signaling in activating gMDSC-restricted inflammasome signaling. Co-culture of intratumoral gMDSCs and KPC CAFs ex vivo revealed strong induction of CAF-intrinsic Il6/Cxcl1 expression, which was dependent in part on CAF-Il1r1 expression and inflammasome activation in gMDSCs. We next used antibody to target ASC-a common downstream adaptor complex inducing inflammasomes-in vivo. Treatment of PKT mice with this anti-ASC antibody significantly attenuated ASC-speck formation in intratumoral gMDSCs as well as CAF-specific Il6/Cxcl1 expression via scRNAseq. Conclusions: These data uncover granulocytic MDSCs as the dominant source of inflammasome activation derived-IL1β in the PDAC TME, which promotes stromal inflammation via iCAF polarization. Therapeutic approaches-such as anti-ASC treatment-targeting gMDSC-intrinsic inflammasome activation may mitigate stromal inflammation and overcome therapeutic resistance in PDAC. Citation Format: Nilesh Deshpande, Anna Bianchi, Iago De Castro Silva, Vanessa Garrido, Siddharth Mehra, Samara Singh, Ifeanyichukwu Ogobuiro, Nagaraj Nagathihalli, Nipun B. Merchant, Jashodeep Datta. Targeting granulocytic MDSC-derived inflammasome activation to overcome stromal inflammation in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C031.

Phase 1 Results of the First-in-Class CXCR1/2 Inhibitor SX-682 in Patients with Hypomethylating Agent Failure Myelodysplastic Syndromes

Background: SX-682 is an oral dual allosteric inhibitor of the CXCR1 and CXCR2 (CXCR1/2) chemokine receptors, a signaling pathway involved in the recruitment of immunosuppressive MDSCs and autocrine growth of disease-initiating leukemic stem cells (LSCs) in AML and myelodysplastic syndromes (MDS). CXCR2 overexpression is predictive of transfusion dependence in MDS. CXCR2 inhibition reduces proliferation of leukemic cell lines and primary MDS/AML patient (pt) samples in vitro and impairs leukemic growth in an in vivo AML animal model. CXCR2 is also involved in normal neutrophil egress from the bone marrow and thus absolute neutrophil count (ANC) serves as a pharmacodynamic (PD) marker for CXCR1/2 inhibition by SX-682. In pts with normal bone marrow, 25-400 mg SX-682 twice-daily (BID) dose-dependently reduces ANC, with maximum ANC reduction (-66% of baseline) obtained at >150 mg BID, consistent with maximum CXCR1/2 inhibition above this dose. ANC rapidly (24 hrs) normalizes with dose-holding. This NHLBI funded trial (HL142389, NCT04245397) explored the safety and efficacy of SX-682 monotherapy in pts with hypomethylating agent (HMA) failure MDS. Methods: This Phase 1 dose-escalation with expansion study treated five cohorts of HMA failure MDS pts with escalating 25-400 mg BID doses of SX-682 in six continuous 28-day cycles with responding pts continuing treatment. Steady-state pharmacokinetics (PK) were obtained. Response assessment was at the end of cycles 1, 3 and 6 and every third cycle thereafter using IWG criteria (Cheson 2006). This interim analysis after dose-escalation to select the expansion dose was pre-specified. Results:17 MDS pts received SX-682 (median 1 prior therapies; range 1-4) with 6 pts in the 200 mg cohort, 3 pts in each of the 25, 50 and 100 cohorts and 2 pts in the 400 mg cohort (Table). Median age was 76, and pts were low (n=1), intermediate-1 (n=10), intermediate-2 (n=5) and high (n=1) risk by the International Prognostic Scoring System (IPSS). All pts were transfusion-dependent and all had failed prior HMA and 24% lenalidomide. Steady-state PK exhibits increasing drug plasma concentration with dose comparable to pts with solid tumors. The ANC PD marker exhibited a dose-dependent decrease from baseline (ΔANC) that plateaued in the 200 and 400 mg dose cohorts consistent with maximum receptor inhibition (Table). Marrow MDSCs and LSCs were reduced after initiation of SX-682. SX-682 was well-tolerated with no maximally tolerated dose and no pt discontinued treatment for adverse events. Treatment-emergent adverse events > grade 3 were most common in the 200 and 400 mg dose cohorts, and those related to neutrophils are consistent with being related to SX-682. Effects on neutrophils reversed on cessation of drug dosing. There was a dose-dependent increase in overall response rate (ORR) from 0% at 25 mg BID to 50% at 200 mg BID consistent with the observed clinical benefit being related to CXCR1/2 inhibition by SX-682 (Table). Four of the five responders had mutant genes (MGs) involving splicing factors (60% of responders; SF3B1, SRSF2) and/or epigenetic regulation (60% of responders; TET2, ASXL1, IDH2). Across all dose cohorts, 8 of 17 (47%) pts had a reduction in marrow blasts after initiating SX-682 (Figure). The 200 mg dose yielded the most rapid and deep reduction in blasts with 2 of 6 pts (33%) achieving a marrow complete remission (mCR) in keeping with this dose providing maximum receptor inhibition. There was hematologic improvement (HI) in 3 pts with one having a durable near-CR response (on SX-682 >500 days) consisting of trilineage HI (hemoglobin > 10 g/dL) and transfusion independence for over 4 months (pt required 16 transfusions in the 7 weeks before starting SX-682 therapy). Based on ANC PD, ORR and marrow blast responses, the 200 mg BID dose was selected for the expansion phase of the trial. Conclusions: SX-682 is a novel therapy in HMA failure MDS with dual activity against LSCs and MDSCs. SX-682 was well tolerated with encouraging efficacy with a biologically effective oral dose selected at 200 mg BID. An expansion cohort in both lower and higher risk MDS pts is ongoing and will additionally include HMA naïve patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PSI-2 Disruption of CXCL12-CXCR4 Axis at Fetal-Maternal Interface During Implantation Alters Ovine Angiogenic Expression at Mid-to-Late Gestation

Abstract Placental dysfunction originates during placental development, specifically the events of placentation. Vascularization is a key component of placentation due to its role in fetal-maternal exchange. Cytokines are regulators of placental development; signaling of chemokine ligand 12 (CXCL12) and its receptor (CXCR4) drives placental vascularization. Utilizing an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface reduces placental vascularization. We hypothesized these negative impacts early in gestation would result in compromised placental development and vascularization at mid to late gestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1x dose (n = 8),1.5X dose (n = 8), or 3X dose (n = 8) or saline (n = 13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 90 and day 135 placental tissues were collected and maternal (caruncle) and fetal (cotyledon) placenta components were separated and analyzed. Gene expression of angiogenic factors were analyzed using Real-time qPCR. Gene expression was significantly altered by AMD3100 treatment for the vascular endothelial growth factor receptors (FLT-1 and KDR) primarily in day 90 placenta contralateral to the corpus luteum. Significant decreases in KDR were seen in 3x compared to control (P &amp;lt; 0.03), 3x compared to 1.5x (P &amp;lt; 0.008), and 3x contralateral compared to ipsilateral (P &amp;lt; 0.01); however, FLT-1 significantly increased (P &amp;lt; 0.02) from control to 1.5x. Day 90 caruncle tissue had significant increases in FLT-1 from control to 3x (0.02), 1.5x to 3x (P &amp;lt; 0.03); however, 1.5x treatment FLT-1 significantly decreased (P &amp;lt; 0.02) ipsilateral to contralateral. Fibroblast growth factor 2 increased in both contralateral 3x compared to control cotyledon (P &amp;lt; 0.03) and caruncle (P &amp;lt; 0.03). Our data underscore the importance of CXCL12-CXCR4 signaling during placentation and provide strong evidence that altering CXCR4-mediated signaling during early placentation induces enduring effects altering vascular development later in gestation. A greater understanding of CXCL12/CXCR4 functions may reveal methods to improve reproductive success and fetal health.

PS-2 Early Intrauterine Suppression of CXCL12-CXCR4 Alters Expression of Placental Inflammatory Profile at Mid-to-Late Gestation

Abstract A proper balance of inflammatory cytokines at the fetal-maternal interface is crucial for successful embryo implantation and placental formation. Chemokine ligand 12 (CXCL12) and receptor CXCR4 are expressed by fetal and maternal placental tissues and implicated in cytokine synthesis. However, mechanisms controlling the axis and placental inflammatory profile are not well characterized. Using an in-vivo sheep model, we previously demonstrated suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface during implantation yielded dysregulated production of inflammatory cytokines in the placenta. Our objective was to determine if disrupting the cytokine profile during implantation alters the placental inflammatory profile at mid-to-late gestation. Day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes to deliver CXCR4 inhibitor (1.5X dose, n = 8; 1X dose, n = 8; 3X dose, n = 8) or saline (n = 13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On days 90 and 135 of gestation, placental tissues were collected; maternal (caruncle) and fetal (cotyledon) placenta components were then separated and analyzed. Real-time qPCR was used to measure gene expression of pro-inflammatory cytokines tumor necrosis factor (TNF), interferon-γ (IFNγ), interleukin-12 (IL12), and anti-inflammatory cytokines interleukin-10 (IL10) and transforming growth factor-β (TGFβ). Gene expression for TGFβ from tissues ipsilateral to the pump on day 90 increased (P &amp;lt; 0.03) in caruncles from 3x treated ewes compared to control ewes while IL10 mRNA decreased (P &amp;lt; 0.02) in cotyledons from 1.5x compared to 3x treatment. Furthermore, analysis of day 135 1x treated ewes showed greater expression (P &amp;lt; 0.03) of IL12 in caruncles ipsilateral compared to contralateral of the pump; meanwhile IL10 showed decreased expression (P &amp;lt; 0.02) in cotyledons ipsilateral compared to contralateral of the pump. High levels of TGFβ, IFNγ (whose production is promoted by increased IL12), and TNF along with decreased levels of IL10 have been associated with preeclampsia in women. The changes in gene expressions of ovine immune cytokines with a disrupted CXCL12-CCR4 axis may be related to preeclamptic events. Ultimately, modulating CXCL12-induced actions is a novel approach to manipulating the fetal-maternal environment when most pregnancy losses occur and may reveal methods to improve reproductive success and fetal health in livestock.

CXCR4 inhibitor, AMD3100, down-regulates PARP1 expression and Synergizes with olaparib causing severe DNA damage in BRCA-proficient triple-negative breast cancer

Poly ADP-ribose polymerase inhibitor (PARPi) treatment is effective in triple-negative breast cancer (TNBC) with BRCA mutation. However, its efficacy in BRCA-proficient TNBC remains unexplored. It is, therefore, an exciting proposition to broaden the indication of PARPi for BRCA-proficient TNBC patients. Chemokine receptor (CXCR4) is a transmembrane G protein-coupled receptor, which is involved in cell migration, proliferation, apoptosis, and damage repair, and it initiates many signalling pathways. Although administration of CXCR4 inhibitor alone is not ideal as a target drug, it can play a strong synergistic role in combination with other drugs. We explored the effect of CXCR4 and PARP1 on tumour cell proliferation, migration, metastasis, and apoptosis in vitro and in vivo and found that a CXCR4 inhibitor, AMD3100, enhanced the anti-tumour effect of PARP1 inhibitor, olaparib, on BRCA-proficient TNBC. When CXCR4 was inhibited and silenced, DNA damage repair and DNA replication fork activity were suppressed by up-regulating caspase-3-mediated increase in PARP1 cleavage; in combination with the inhibition of PARP1, AMD3100 resulted in the accumulation of fatal DNA damage and induction of apoptosis. This combination regimen can be effective against BRCA-proficient TNBC.

PSI-9 Disrupting Cxcl12-Cxcr4 Chemokine Axis at the Fetal-Maternal Interface During Implantation Results in Altered Expression of Angiogenic and Insulin-Like Growth Factors in Ovine Placenta Midgestation

Abstract Placental dysfunction is a fundamental cause of intrauterine growth restriction, and fetal morbidity and mortality in livestock. Though causes of placental dysfunction are not well characterized, origin of disease transpires during placental development (placentation). The C-X-C motif chemokine ligand 12 (CXCL12) working through its receptor CXCR4 is implicated in placentation but precise roles of this axis are unclear. Using an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface reduces placental vascularization. We hypothesized these negative impacts early in gestation would result in compromised placental development and vascularization at midgestation. To test, onday 12 post-breeding, osmotic pumps were surgically installed in 22 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1.5X dose, (n=8) or 3X dose (n=8) or saline (n=6) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On day 90 placental tissues were collected and maternal and fetal placenta components separated and analyzed. The objectives were to determine if suppressing CXCL12-CXCR4 actions at the fetal-maternal interface during implantation results in reduced placental and vascular development at midgestation. Gene expression for angiogenic factors and members of the insulin-like growth factor family were analyzed using Real-time qPCR. Of the targets tested, mRNA expression was significantly altered by AMD3100 treatment for the vascular endothelial growth factor receptors (FLT-1 and KDR) primarily in maternal placenta. Expression of insulin-like growth factors 1 and 2, IGF-binding protein 3, and placental lactogen were significantly altered in fetal placenta with AMD3100 treatment compared to control. Our preliminary data underscore the importance of CXCL12-CXCR4 signaling during placentation and provide strong evidence that altering CXCL12-mediated signaling during early placentation induces enduring placental effects manifesting later in gestation. A greater understanding of CXCL12/CXCR4 functions may reveal methods to improve reproductive success and fetal health.

PSII-A-11 Suppressing the Cxcl12-Cxcr4 Chemokine Axis at the Fetal-Maternal Interface During Implantation Results in Altered Expression of Inflammatory Cytokines in Ovine Placenta at Midgestation

Abstract A proper balance of pro and anti-inflammatory cytokines at the fetal-maternal interface is crucial for successful embryo implantation and placental formation (placentation) during early gestation. However, mechanisms controlling this inflammatory profile are not well characterized. The C-X-C motif chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal placental tissues and implicated in cytokine synthesis; roles of this axis during placentation are unclear. Using an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface during implantation results in dysregulated production of inflammatory cytokines in the placenta. The objective of the current study was to determine if the disrupted cytokine profile from suppressing CXCL12-CXCR4 signaling early in gestation would result in altered placental inflammatory profile at midgestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1X dose, (n=8), 1.5X, (n=8), or 3X, (n=8) or saline (n=13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On days 90 and 135, placental tissues were collected, and maternal and fetal placenta components separated and analyzed. Gene expression for select cytokines were analyzed in the placenta using Real-time qPCR. On day 90, mRNA expression was significantly altered by AMD3100 treatment for tumor necrosis factor (TNF), interleukin 12 (IL-12), and transforming growth factor beta (TGFB) in fetal placenta. Expression of TGFB in fetal placenta was also altered by AMD3100 treatment compared to control on day 135 whereas IL-12 expression was significantly altered in maternal placenta on day 135. In conclusion, modulating CXCL12-induced actions is a novel approach to manipulating the fetal-maternal environment when most pregnancy losses occur. Increasing our understanding of CXCL12-induced actions controlling the placental inflammatory environment will hopefully reveal methods to improve reproductive success and fetal health in livestock.

Loss of Cxcr2 in Myeloid Cells Promotes Tumour Progression and T Cell Infiltration in Invasive Bladder Cancer.

CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2 flox/flox ). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status.

Bronchiectasis: A pulmonary disease with systemic consequences.

Bronchiectasis: A pulmonary disease with systemic consequences.

CXCL12/CXCR4 Axis Drives the Chemotaxis and Differentiation of B Cells in Bullous Pemphigoid

Bullous pemphigoid (BP) is an autoimmune bullous skin disease characterized by autoantibodies against the hemidesmosomal proteins in the skin and mucous membranes. The efficiency of B-cell‒targeting biologics in BP indicates the important role of B cells in its pathogenesis. However, abnormal B-cell migration and differentiation in BP require further elucidation. We showed that the number of antibody-secreting cells increased in the circulation and skin lesions of patients with BP and was correlated with disease severity. Bulk RNA sequencing of the peripheral B cells identified 171 upregulated and 408 downregulated genes in patients with BP compared with those in healthy controls, among which CXCR4 was significantly upregulated. Notably, CXCR4+ B cells were enriched in BP skin lesions and exhibited antibody-secreting cell characteristics. Correspondingly, an elevated level of CXCL12, the CXCR4 ligand, was detected in the blister fluid and serum of patients with BP, mediating the chemotaxis and accumulation of CXCR4+ B cells to BP skin lesions. Moreover, CXCL12 activated the transcription factor c-Myc, thus promoting B-cell differentiation into antibody-secreting cells and facilitating autoantibody production, which was blocked by CXCR4 inhibitor invitro. Collectively, our study reveals that the CXCL12/CXCR4 axis plays a pathogenic role in modulating B-cell trafficking and differentiation, thus targeting CXCR4 represents a potential strategy for treating BP and other autoimmune diseases.