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Protein folding complexes are a vital link between the expression of tumor-promoting oncogenes and oncogenic cancer behavior known as the hallmarks of cancer. Of these, the eukaryotic type II chaperonin, Chaperonin-Containing TCP-1 (CCT or TRiC) folds many of the oncoproteins (e.g., KRAS, MYC, CDKs, STAT3, etc.) that drive cancer growth and invasion. CCT is a complex machine composed of eight subunits, each encoded by a unique gene (cct1-8), which folds proteins in an ATP-dependent fashion within an inner chamber. Previous work in breast cancer revealed that the CCT2 subunit was highly expressed in breast tumor tissues and correlated with advanced breast cancer stage, metastasis, and decreased patient survival. To determine the pattern of CCT2 expression across multiple cancers in comparison to normal tissues, we interrogated the UCSC Xena database to compare cohorts, GTEx (normal tissues), TCGA (adult cancerous and normal tissues), and TARGET (pediatric cancerous and normal tissues). Tumor specimens from adult TCGA expressed significantly higher levels of CCT2 than GTEx, and pediatric TARGET samples had significantly higher expression levels of CCT2 than TCGA and normal samples. Histological detection of CCT2 in multiple cancer tissues was increased (e.g., pediatric cancers and sarcomas being among the highest) compared to cancer-adjacent tissues or normal tissues and supported the bioinformatics data. Manipulating CCT2 levels in individual breast, neuroblastoma, and prostate cancer cells revealed the strategic importance of this chaperonin subunit in supporting the metastatic cancer phenotype. Exogenous expression of CCT2 promoted uncontrolled and anchorage-independent growth and migration of cancer cells, which was reversed upon depletion of the subunit, causing cancer cell death. Moreover, conditioned media from cancer cells exogenously expressing CCT2 also drove the migration and growth of cancer cells. Exosomes isolated from this media contained high levels of CCT2 mRNA, further demonstrating the metastasis-promoting potential of this chaperonin subunit. The feasibility of using CCT2 as a biomarker to detect cancer progression was confirmed using antibodies to detect CCT2 in the identification of circulating tumor cells using the CellSearch System. These results provide validation to further develop the use of CCT2 as a diagnostic marker for cancer progression and metastasis and as a promising therapeutic target for new drug development. Citation Format: Annette R. Khaled, James Velazquez, Lam Truong, Colten Frank, Carolyn Dang, Amanda Cox, Heba Ghozlan, Eunkyung Lee, Amr S. Khaled, Priya K. Gopalan. Protein folding chaperonin as biological indicator for cancer progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2463.

Research shows providing cancer patients with adequate information has many benefits, but how do nurse practitioners know whether the initial consultation meets the information needs of their patients? Furthermore, how can the initial consultation be improved?A low-cost continuous quality improvement process centered on decreasing distress and increasing information satisfaction was piloted to determine its effectiveness and feasibility.Immediately before and after an initial consultation with a breast cancer surgeon, 59 women completed a questionnaire to measure distress and specific problems they were having. They then completed a questionnaire to measure information satisfaction. Pre-post changes in the distress score and number of problems were analyzed, as was information satisfaction. Feasibility was qualitatively examined.For the study sample, pre-post median distress scores decreased significantly (from 5 to 3, Chi-square = 5.73,This effective and feasible process may help the nurse practitioner continuously improve the initial consultation process.(a) the initial breast cancer consultation is important, (b) a novel process for improving the initial breast cancer consultation is proposed, and (c) this feasible, low-cost process should be embedded into normal practice operations.

Aberrant activation of CDK4/6 kinase is the most common somatic event in non-small cell lung cancer (NSCLC). Palbociclib is a highly specific CDK4/6 inhibitor shown to inhibit cell cycle progression and promote cellular senescence. We conducted a phase 2 clinical trial of palbociclib in 19 previously-treated patients with advanced NSCLC. Only patients with p16-null staining by immunohistochemistry and documented tumor progression were eligible. The primary endpoint was tumor response rate. Palbociclib therapy alone was well-tolerated. Of 16 evaluable patients who received > 1 month of therapy, there were no objective responses. However, 8 patients (50%) with previously progressive NSCLC had stable disease (SD) lasting a range of 4-10.5 months. Median overall survival (OS) for all cases was 5.1 months, and median overall survival for the subset of patients with SD was 16.6 months. We also performed preclinical testing of palbociclib in combination with 13 different targeted or cytotoxic chemotherapeutic agents using a cell viability assay. Only the combination of palbociclib and mTOR inhibitors resulted in synergistic growth inhibition, particularly in tumors carrying RAS mutations. Our findings warrant further clinical investigation of the combination of palbociclib and mTOR inhibitors, especially in patients carrying activated RAS mutations.

Chaperonin-containing TCP-1 (CCT or TRiC) is a multi-subunit complex that folds many of the proteins essential for cancer development. CCT is expressed in diverse cancers and could be an ideal therapeutic target if not for the fact that the complex is encoded by eight distinct genes, complicating the development of inhibitors. Few definitive studies addressed the role of specific subunits in promoting the chaperonin’s function in cancer. To this end, we investigated the activity of CCT2 (CCTβ) by overexpressing or depleting the subunit in breast epithelial and breast cancer cells. We found that increasing total CCT2 in cells by 1.3-1.8-fold using a lentiviral system, also caused CCT3, CCT4, and CCT5 levels to increase. Likewise, silencing cct2 gene expression by ~50% caused other CCT subunits to decrease. Cells expressing CCT2 were more invasive and had a higher proliferative index. CCT2 depletion in a syngeneic murine model of triple negative breast cancer (TNBC) prevented tumor growth. These results indicate that the CCT2 subunit is integral to the activity of the chaperonin and is needed for tumorigenesis. Hence CCT2 could be a viable target for therapeutic development in breast and other cancers.

The purpose of this quality improvement study was to improve physician documentation of distress in medical records of hematology/oncology veteran patients at the Malcolm Randall Veteran Affairs (VA) Medical Center hematology/oncology fellows’ clinic in Gainesville, Florida. Before this intervention, the VA hematology/oncology fellows were not documenting patient distress in medical records. The quality improvement intervention was executed through the use of Plan-Do-Study-Act (PDSA) cycles with an ultimate goal of 50% documentation rate. Physician charts were audited to investigate official documentation of distress in patient charts. Physician documentation of distress was 14% in the first PDSA cycle, 21% in the second PDSA cycle, and 36% in the third PDSA cycle. Additional data on distress in hematology/oncology veteran patients were collected using the National Comprehensive Cancer Network Distress Thermometer and Problem List for Patients. Analysis of findings indicated that 42% of 88 patients experienced distress. Findings also suggest that hematology/oncology veteran patients experience specific sources of distress, notably fatigue and pain. These patients have presumably undergone unique experiences that can result in distress that providers should follow-up with in medical charts. Although this intervention has proven challenging to fully implement, standardizing patient distress in patient medical records has the potential to improve the quality of care provided by hematology/oncology physicians.

PURPOSE To evaluate the rate and risk factors of isolated leptomeningeal progression in sinonasal carcinomas. METHODS We retrospectively reviewed imaging and clinical records to determine progression patterns, and estimated rates using the Kaplan-Meier method. We evaluated risk factors using proportional hazard regression. RESULTS We analyzed 120 patients who received adjuvant or primary radiotherapy for sinonasal carcinomas. Most patients had T4 disease (68%) and underwent surgery (84%) and chemotherapy (72%). Twenty-seven (23%) patients developed distant metastases (DM), including 20 (17%) with isolated DMs. Leptomeningeal progression was the most common site of isolated DMs (n = 9; 45%) with an average disease-free interval of 1.2 years (0.1-4.3 years). High-grade histology (P = 0.0003), intracranial invasion (P

We conducted a retrospective study of stereotactic ablative radiotherapy (SABR) for 94 patients with non-small-cell lung cancer at our institution. The patients were treated with either 50 Gy in five treatments or 48 Gy in four treatments, corresponding to biologically effective doses (BED) of 100 Gy or 105.6 Gy, respectively. The results demonstrate that, with relatively low BEDs, we can achieve excellent local control with minimal toxicity.

Introduction Pain is one of the most burdensome symptoms associated with cancer and its treatment, and opioids are the cornerstone of pain management. Opioid therapy is empirically selected, and patients often require adjustments in therapy to effectively alleviate pain or ameliorate adverse drug effects that interfere with quality of life. There are data suggesting CYP2D6 genotype may contribute to inter-patient variability in response to opioids through its effects on opioid metabolism. Therefore, we aim to determine if CYP2D6 genotype-guided opioid prescribing results in greater reductions in pain and symptom severity and interference with daily living compared to a conventional prescribing approach in patients with cancer. Methods Patients with solid tumors with metastasis and a self-reported pain score ≥ 4/10 are eligible for enrollment and randomized to a genotype-guided or conventional pain management strategy. For patients in the genotype-guided arm, CYP2D6 genotype information is integrated into opioid prescribing decisions. Patients are asked to complete questionnaires regarding their pain, symptoms, and quality of life at baseline and 2, 4, 6, and 8 weeks after enrollment. The primary endpoint is differential change in pain severity by treatment strategy (genotype-guided versus conventional pain management). Secondary endpoints include change in pain and symptom interference with daily living. Conclusion Pharmacogenetic-guided opioid selection for cancer pain management has potential clinical utility, but current evidence is limited to retrospective and observational studies. Precision Medicine Guided Treatment for Cancer Pain is a pragmatic clinical trial that seeks to determine the utility of CYP2D6 genotype-guided opioid prescribing in patients with cancer.

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are less clear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect associated with induction of toll-like receptor (TLR) expression and signaling. The G-CSF-mediated expansion of HSCs is reduced in mice lacking TLR2, TLR4 or the TLR signaling adaptor MyD88. Induction of HSC quiescence is abrogated in mice lacking MyD88 or in mice treated with antibiotics to suppress intestinal flora. Finally, loss of TLR4 or germ free conditions mitigates the G-CSF-mediated HSC repopulating defect. These data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling.

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Neutrophils form CD18-dependent adhesions to endothelial cells at sites of inflammation. This phenomenon was investigated under conditions of flow in vitro using isolated human neutrophils and monolayers of HUVEC. The efficiency of conversion of neutrophil rolling to stable adhesion in this model was >95%. Neither anti-CD11a nor anti-CD11b antibodies significantly altered the extent of this conversion, but a combination of both antibodies inhibited the arrest of rolling neutrophils by >95%. The efficiency of transendothelial migration of arrested neutrophils was >90%, and the site of transmigration was typically

Background Having gained subspecialty certification in 2003, the field of psychosomatic medicine (PM) addresses the mental health needs of individuals who suffer from general medical conditions. The rising prevalence of chronic illness, along with trends in medical delivery toward more collaborative models of care, underscores the value of recruitment to PM specialty programs. Objectives To foster interest and education in PM, we have developed and implemented a Psychosomatic Medicine Interest Group for trainees within a psychiatry residency program. Results Participants have found the Psychosomatic Medicine Interest Group to be an enjoyable experience that has improved their clinical practice and interest in PM. Conclusion The Psychosomatic Medicine Interest Group has also been a successful vehicle to enhance clinical knowledge and mentoring opportunities during training, while bolstering residents’ desire to pursue a career in PM.

Background: Sunitinib is an oral multitargeted tyrosine kinase inhibitor, with single-agent activity in non-small cell lung cancer (NSCLC). Resistance to tyrosine kinase inhibitor therapy is mediated by the mammalian target of rapamycin (mTOR) pathway, and may be reversed by using mTOR inhibitors. Methods: We performed a phase I study evaluating the combination of sunitinib and rapamycin in patients with advanced NSCLC. Results: Nineteen patients were enrolled in the study. The dose-limiting toxicities included infection, pneumonia, diarrhea/dehydration and treatment delay due to thrombocytopenia in 1 patient each. Sunitinib 25 mg orally daily and rapamycin 2 mg orally daily with 4 weeks on and 2 weeks off therapy were determined to be the maximum tolerated dose. No objective responses were noted, and 6 patients had stable disease as a best response. Conclusion: The combination of sunitinib and rapamycin is well-tolerated and warrants further investigation in the phase II setting.

Neutrophils are a major component of the innate immune response. Their homeostasis is maintained, in part, by the regulated release of neutrophils from the bone marrow. Constitutive expression of the chemokine CXCL12 by bone marrow stromal cells provides a key retention signal for neutrophils in the bone marrow through activation of its receptor, CXCR4. Attenuation of CXCR4 signaling leads to entry of neutrophils into the circulation through unknown mechanisms. We investigated the role of CXCR2-binding ELR+ chemokines in neutrophil trafficking using mouse mixed bone marrow chimeras reconstituted with Cxcr2(-/-) and WT cells. In this context, neutrophils lacking CXCR2 were preferentially retained in the bone marrow, a phenotype resembling the congenital disorder myelokathexis, which is characterized by chronic neutropenia. Additionally, transient disruption of CXCR4 failed to mobilize Cxcr2(-/-) neutrophils. However, neutrophils lacking both CXCR2 and CXCR4 displayed constitutive mobilization, showing that CXCR4 plays a dominant role in neutrophil trafficking. With regard to CXCR2 ligands, bone marrow endothelial cells and osteoblasts constitutively expressed the ELR+ chemokines CXCL1 and CXCL2, and CXCL2 expression was induced in endothelial cells during G-CSF-induced neutrophil mobilization. Collectively, these data suggest that CXCR2 signaling is a second chemokine axis that interacts antagonistically with CXCR4 to regulate neutrophil release from the bone marrow.

8077 Background: The Retinoblastoma pathway is targeted for mutational or epigenetic inactivation in more than 70% of NSCLC. The most common event is loss of CDKN2A expression (p16 protein), usuall...

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte-colony stimulating factor (G-CSF) is often induced with infection and plays a key role in the stress granulopoiesis response. However, its effects on HSCs are unclear. Herein, we show that treatment with G-CSF induces expansion and increased quiescence of phenotypic HSCs, but causes a marked, cell-autonomous HSC repopulating defect. RNA profiling and flow cytometry studies of HSCs from G-CSF treated mice show that multiple toll- like receptors (TLRs) are upregulated in HSCs upon G-CSF treatment, and gene set enrichment analysis shows enhancement of TLR signaling in G-CSF-treated HSCs. G-CSF-induced expansion of phenotypic HSCs is reduced in mice lacking the TLR signaling adaptors MyD88 or Trif, and the induction of quiescence is abrogated in mice lacking these adaptors. Furthermore, loss of TLR4 mitigates the G-CSF-mediated HSC repopulating defect. Interestingly, baseline HSC function is also dependent on TLR signaling. We show that HSC long-term repopulating activity is enhanced in Tlr4-/- and MyD88-/- mice, but not Trif-/- mice. One potential source of TLR ligands affecting HSC function in the bone marrow is the gut microbiota. Indeed, we show that in mice treated with antibiotics to suppress intestinal flora, G-CSF induced HSC quiescence and hematopoietic progenitor mobilization are attenuated. Moreover, in germ free mice, HSC long-term repopulating activity is enhanced. Collectively these data suggest that low level TLR agonist production by commensal flora contributes to the regulation of HSC function and that G-CSF negatively regulates HSCs, in part, by enhancing TLR signaling. Our finding of enhanced TLR signaling upon G-CSF treatment, and the mitigation of G-CSF’s effects in mice deficient for TLR signaling or commensal organisms, suggest that TLR antagonists and/or agonists may ultimately be used clinically to enhance engraftment following bone marrow transplantation or applied toward the treatment of patients with bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

The p16 protein plays a critical role in the regulation of the retinoblastoma (RB) pathway, which inhibits cell growth through cell cycle regulation. The p16 gene, CDKN2a, is inactivated in 70% of NSCLC tumors, resulting in unregulated activation of CDK 4/6, and consequently increased cell cycling. The drug PD 0332991 (PD) is a highly specific inhibitor of CDK4/6. Western blots of p16-deficient NSCLC cell lines have confirmed a decrease in phosphorylated RB with PD-treated cells compared to untreated cells. We have observed a 34% decrease in viability by an MTS assay, and a 30% increase in cell senescence by a β-galactosidase assay in PD-treated p16-deficient cell lines compared to untreated cells. Based on these data, we are currently conducting a single-arm phase II clinical trial of single-agent PD 0332991 in patients with advanced NSCLC who have failed at least one prior chemotherapy regimen. We hypothesized that a combination of PD with other targeted therapies, acting on the same and intersecting pathways, would result in increased tumor cell death. Using MTS viability assays, we tested PD with and without other inhibitors in p16-deficient NSCLC cell lines. These inhibitors were: 1) the demethylating agent, decitabine, since the p16 gene, CDKN2a, is most often inactivated by hypermethylation (PD 66.1%, decitabine 49.7%, combination 80.2% viability); 2) the MEK inhibitor, AZD 6244, based on a report of a potential synthetic lethal interaction between K-Ras and CDK4 (PD 66.1%, AZD 6244 90.1%, combination 72.0% viability); 3) the CDK1/2/9 inhibitor, AZD 5438, since CDK 1 and 2 are thought to play a role in resistance to CDK 4/6 inhibition (PD 66.1%, AZD 5438 76.3%, combination 96.6% viability), and 4) the mTOR inhibitor, everolimus, since the mTOR pathway can upregulate cyclin D1, which complexes with CDK4/6 in the RB pathway (PD 67.3%, everolimus 65.1%, combination 46.4% viability, p Citation Format: Priya K. Gopalan, Andres Gordillo-Villegas, Maria Zajac-Kaye, Frederic J. Kaye. Inhibitory effect of the CDK4/6 inhibitor, PD 0332991, is enhanced by mTOR inhibition in Non-Small Cell Lung Cancer (NSCLC). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2013-693

This unit describes a hydrodynamic assay to study the relative importance of various receptor/ligand interactions in cell-cell and cell-substrate adhesion and to quantitate the strength of their binding. The basic protocol describes how to assemble the single-chamber flow system with the substrate, add the cells in suspension, and record the experiment on videotape. Alternate protocols present assays to determine how monoclonal antibodies and stimulating and inhibiting agents affect the substrate and the perfusing cells in suspension. Another alternate protocol details the use of the double-chamber flow system. Support protocols describe how to construct the single- and double-chamber flow systems and how to analyze the data from an experiment. Recording and analyzing the flow experiment requires the use of video equipment and, optionally, a computer and imaging software.

Abstract 2356 Kruppel like factor 7 (KLF7) expression is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia (Flotho, et al; Blood 2007). In addition, KLF7 overexpression is associated with Imatinib-resistant CML (Cammarata, et al; Clinical Leukemia 2007). The kruppel like factor (KLF) family of transcription factors are involved in regulating cellular growth and differentiation in multiple tissue types. KLF7 is important for neurogenesis, and mice lacking KLF7 die perinatally with severe neurologic defects (Laub, et al; Mol Cell Biol 2005). While no specific role for KLF7 in hematopoiesis has been previously reported, loss of the closely related family member KLF6 is associated with defective blood cell production (Matsumoto, et al; Blood 2006), and other KLF family members are involved in multiple aspects of hematopoiesis. Targets of KLF7 include known regulators of hematopoietic stem and progenitor cell (HSPC) function including TRKA, Cebp/a, and CDKN1A (p21). Normal HSPCs appear to have a low level of KLF7 expression based on RNA expression profiling of populations enriched for these cells. Given these findings, we hypothesized that KLF7 may play a role in regulating normal HSPC function, and may contribute to leukemogenesis or resistance to therapy. To test this hypothesis, we first analyzed the effect of the loss of KLF7 on hematopoiesis. Specifically, we generated Klf7−/− fetal liver chimeras and characterized their hematopoiesis. Long-term multilineage engraftment of Klf7−/− cells was comparable to control cells. Moreover, HSC self-renewal, as assessed by serial transplantation was not effected by the loss of KLF7. To model the effect of KLF7 overexpression on HSPC function, we generated retroviral and lentiviral vectors that express KLF7. KLF7 expression in wild type bone marrow cells transduced with KLF7 retrovirus was increased approximately 10-fold. Overexpression of KLF7 was associated with a marked suppression of myeloid progenitor cell growth, as assessed using colony-forming cell assays. Relative to the initial transduction efficiency, the number of myeloid colonies produced from KLF7-transduced cells compared to vector-alone transduced cells was reduced 5.7 ± 1.9 fold. We next assessed short- and long-term engraftment of KLF7-transduced cells by bone marrow transplantation. In experiments using bone marrow cells transduced with high efficiency (≥ 60% transduced cells), overexpression of KLF7 resulted in impaired radioprotection. Whereas all (12 of 12) recipients transplanted with control transduced cells survived, only 42% (5 out of 12) of recipients of KLF7 transduced cells survived more than two weeks after transplantation (P < 0.003). When these experiments were performed with a reduced multiplicity of infection to achieve a lower transduction efficiency, all recipient mice survived at least 3 months. Whereas control-transduced cells were readily detected at near input levels (on average, 40% of nucleated blood cells), minimal contribution of KLF7-transduced cells was observed in all lineages except T cells. Interestingly, KLF7-transduced T cells were present at near input levels. In summary, our show that KLF7 is not required for normal HSPC function. However, overexpression of KLF7 leads to a marked suppression of the short- and long-term repopulating activity of HSPC with the exception cells in the T cell lineage. Whether KLF7 expression contributes to T cell leukemogenesis through suppression of other hematopoietic lineages will require further study. Disclosures: No relevant conflicts of interest to declare.

e21131 Background: Chronic immunosuppression, required to prevent organ rejection in transplant recipients, is associated with an elevated risk for lymphoid and solid tumors. While cutaneous cancer...

Abstract 235 Truncation mutations of CXCR4 that cause increased receptor signaling are responsible for most cases of WHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome, which is characterized by the retention of mature neutrophils in the bone marrow despite peripheral neutropenia. This observation and others have established CXCR4 as a key regulator of neutrophil release from the bone marrow. However, it is unclear how modulation of neutrophil CXCR4 signaling is linked to their migration toward the vascular endothelium and subsequent entry into the circulation. Therefore, the question of whether neutrophil egress from the bone marrow is a passive, random process or actively directed and what (if any) signals regulate it remains unanswered. We recently analyzed a myelokathexis pedigree and discovered homozygous, loss-of-function mutations in CXCR2. Based on these observations, we developed a “tug-of-war” model in which opposing chemokine gradients, specifically release-inducing CXCR2 signals and retention-promoting CXCR4 signals, act antagonistically to regulate neutrophil release from the bone marrow. To test this model, we analyzed neutrophil trafficking in CXCR2−/− mice. These mice have a well-characterized defect in neutrophil emigration from the blood to sites of inflammation, leading to chronic subclinical infection and the systemic release of cytokines that stimulate granulopoiesis. To circumvent these potentially confounding effects, we generated mixed bone marrow chimeras reconstituted with a 1:1 ratio of wild-type and CXCR2−/− bone marrow. As expected, approximately 50% (46 ± 4%) of circulating B cells were derived from CXCR2−/− cells. In contrast, a significant decrease in the proportion of CXCR2−/− neutrophils in the blood was observed (23 ± 4%; P < 0.001). Consistent with a myelokathexis phenotype, there was a relative accumulation of mature CXCR2−/− neutrophils in the bone marrow (47 ± 9% of Gr-1hi SSChi cells in the bone marrow were derived from CXCR2−/− cells; P < 0.01). Neutrophil trafficking from the bone marrow was estimated by calculating the percentage of neutrophils in the blood out of the total amount in the blood and bone marrow (neutrophil distribution index or NDI). We estimated that 1.3 ± 0.2% of wild-type neutrophils were in the blood, but the percentage of CXCR2−/− neutrophils in the blood was reduced to 0.4 ± 0.1% (P < 0.01). These data provide genetic evidence that CXCR2 signals promote neutrophil release from the bone marrow in a cell-autonomous manner. To explore the epistatic relationship of CXCR2 and CXCR4 signals to neutrophil trafficking, the neutrophil response to AMD3100, a small molecule CXCR4 antagonist, was examined in the CXCR2−/− mixed chimeras. Consistent with previous reports, treatment with AMD3100 resulted in a 5.2 ± 0.7-fold increase in wild-type neutrophils in the blood one hour after administration. In contrast, AMD3100 induced release of CXCR2−/− neutrophils was impaired, with only a 2.8 ± 0.3-fold increase observed (P < 0.05). G-CSF treatment is thought to induce neutrophil release through disruption of CXCR4 signaling. Thus, we next characterized the neutrophil response to 5 days of G-CSF treatment. Wild-type neutrophils displayed a shift from the bone marrow to the blood, with an NDI of 5.0 ± 0.4%. The number of CXCR2−/−neutrophils in the blood increased after treatment, but the percentage in the blood (2.5 ± 0.7%) was less than wild-type (P < 0.05). These data show that maximal neutrophil release requires the coordinated regulation of CXCR2 and CXCR4 signals. Studies are underway to assess neutrophil trafficking of CXCR4−/− × CXCR2−/− neutrophils. The tug-of-war model of neutrophil trafficking in the bone marrow predicts that CXCR2 ligands will be highly expressed in bone marrow endothelial cells or other cells closely associated with the endothelium. To test this prediction, endothelial cells (CD45− Ter119− CD31+) were sorted from the bone marrow of wild-type mice at baseline or after 5 days of G-CSF treatment. RNA expression profiling showed constitutive high level expression of the CXCR2 ligands CXCL1 and CXCL2. Moreover, expression of CXCL2 was significantly induced after G-CSF treatment. Chemokine expression was confirmed by real time RT-PCR and ELISA. Taken together, our data suggest that CXCR2 signaling is a second chemokine pathway that, in coordination with CXCR4, controls neutrophil release from the bone marrow. Disclosures: No relevant conflicts of interest to declare.

The bone marrow microenvironment plays a key role in regulating hematopoietic stem cell (HSC) function. In particular, bone marrow stromal signals contribute to the maintenance of HSC quiescence, a property that is thought to be associated with long-term repopulating activity. We previously reported that G-CSF treatment disrupts the osteoblast niche by inducing osteoblast apoptosis and inhibiting osteoblast differentiation. In this altered bone marrow microenvironment, we also showed that the number of HSCs in the bone marrow after G-CSF treatment (as defined by CD34− Kit+ Sca+ lineage-cells or CD150+ CD48− CD41− lineage-[SLAM] cells) was unchanged and that the HSCs were more quiescent than HSCs from untreated mice. However, despite the quiescent phenotype, there was a marked loss of HSC long-term repopulating activity. To define mechanisms for this phenotype, we first asked whether G-CSF acts directly on HSCs to inhibit their long-term repopulating activity. Bone marrow chimeras containing wild type and G-CSFR−/− cells were established and treated with G-CSF. The contribution of G-CSFR−/− cells to hematopoiesis remained stable for at least 3 months after G-CSF treatment, demonstrating that the effects of G-CSF on HSC function are not direct. We next performed RNA expression profiling on sorted SLAM cells, a cell population highly enriched for HSCs. These data showed that expression of Cdkn1a (p21cip1/waf1) was increased in HSCs harvested from G-CSF treated mice. To define the contribution of Cdkn1a to HSC quiescence and loss of repopulating activity following treatment with G-CSF, Cdkn1a−/− mice (inbred on a C57BL/6 background) were studied. Wild-type or Cdkn1a−/− mice were treated with G-CSF for 7 days and pulse labeled with bromo-deoxyuridine (BrdU), and the percentage of SLAM cells that labeled with BrdU was determined. Consistent with our previous observations, treatment of wild-type mice with G-CSF resulted in a significant decrease in the percentage of BrdU+ SLAM cells in the bone marrow. In contrast, in Cdkn1a−/− mice, no change in the percentage of BrdU+ SLAM cells after G-CSF treatment was observed [10.08 ± 2.26% (untreated); 10.96 ± 2.80% (G-CSF treated); p = NS]. To assess HSC function, competitive repopulation assays were performed using untreated or G-CSF treated bone marrow from wild type or Cdkn1a−/− mice. Surprisingly, G-CSF had a similar deleterious effect on HSC repopulating activity in both wild type and Cdkn1a−/− mice. Collectively, these data show G-CSF treatment, possibly through disruption of the osteoblast niche, induces HSC quiescence and loss of long-term repopulating activity. HSC quiescence, but not loss of repopulating activity, is dependent upon Cdkn1a−/−. The mechanisms by which G-CSF treatment results in a loss of HSC function are under investigation.

There is evidence that hematopoietic stem cells (HSC) are physically localized to specialized areas in the bone marrow termed the vascular and osteoblast niches. It is not clear if there are differences in the capacity of these niches to support HSC function. We and others previously showed that G-CSF treatment suppresses both osteoblast number and function, effectively eliminating the osteoblast niche. In contrast, G-CSF treatment has no apparent effect on the microvasculature in the bone marrow, suggesting that the vascular niche is intact. In this study, we utilized this system to assess the capacity of each niche to support HSC function. We previously reported that the competitive repopulation capacity of bone marrow isolated from G-CSF treated mice is markedly reduced. This is not due to a simple loss of HSC in the bone marrow, as the number of HSC, phenotypically defined as lineage-CD41-CD48-CD150+ (SLAM) or lineage-Kit+Sca+CD34- cells, was comparable to control mice. Moreover, the long-term repopulating activity of sorted SLAM cells from G-CSF treated mice was reduced. This repopulating defect is not secondary to impaired homing to the bone marrow, as direct intrafemoral injection of G-CSF treated bone marrow cells failed to rescue the engraftment defect. Since G-CSF is able to stimulate HSC proliferation, we predicted that the defect in repopulating activity might be secondary to loss of HSC quiescence. Contrary to our prediction, the percentage of quiescent HSC in the bone marrow was actually increased in G-CSF treated mice. Whereas 28.0 ± 3.4% of control SLAM cells were labeled after treatment with BrdU for 48 hours, only 7.5 ± 0.8% of SLAM cells isolated from G-CSF mice were labeled (p < 0.008). Moreover, the percentage of SLAM cells in G0, as determined by Hoechst and pyronin staining, was increased in G-CSF treated mice (80.3 ± 5.0% versus 65.5 ± 6.8% in untreated mice, p=0.104). To elucidate the molecular mechanisms by which disruption of the osteoblast niche leads to a loss of HSC activity, we performed RNA profiling experiments on SLAM cells sorted from G-CSF or saline-treated mice. Consistent with the quiescent phenotype, a significant increase in the expression of the cell cycle inhibitor, Cdkn1a (p21waf1), was observed in G-CSF treated SLAM cells. Collectively, these data show that the osteoblast and vascular niches are not functionally redundant and suggest that it is the osteoblast niche that is key to maintaining long-term repopulating activity of HSC.

Accumulating evidence suggests that osteoblast-lineage cells play a key role in supporting hematopoiesis, providing signals that maintain the normal function of hematopoietic stem cells (HSC). We and others previously showed that treatment with G-CSF reduces the number and activity of osteoblasts in the bone marrow. Here we confirm these findings using transgenic mice expressing GFP in osteoblast lineage cells under control of a 2.3 kb fragment of the col1a1 promoter. Analysis of these mice confirmed that G-CSF treatment reduced the number of osteoblasts 2.9-fold compared to control mice (n=4, p