Priya K. Gopalan

CXCR2 and CXCR4 antagonistically regulate neutrophil trafficking from murine bone marrow

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.

G-CSF regulates hematopoietic stem cell activity, in part, through activation of Toll-like receptor signaling.

Recent studies demonstrate that inflammatory signals regulate hematopoietic stem cells (HSCs). Granulocyte colony-stimulating factor (G-CSF) is often induced with infection and has 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.

Kruppel-like factor 7 overexpression suppresses hematopoietic stem and progenitor cell function

Increased expression of Kruppel-like factor 7 (KLF7) is an independent predictor of poor outcome in pediatric acute lymphoblastic leukemia. The contribution of KLF7 to hematopoiesis has not been previously described. Herein, we characterized the effect on murine hematopoiesis of the loss of KLF7 and enforced expression of KLF7. Long-term multilineage engraftment of Klf7(-/-) cells was comparable with control cells, and self-renewal, as assessed by serial transplantation, was not affected. Enforced expression of KLF7 results in a marked suppression of myeloid progenitor cell growth and a loss of short- and long-term repopulating activity. Interestingly, enforced expression of KLF7, although resulting in multilineage growth suppression that extended to hematopoietic stem cells and common lymphoid progenitors, spared T cells and enhanced the survival of early thymocytes. RNA expression profiling of KLF7-overexpressing hematopoietic progenitors identified several potential target genes mediating these effects. Notably, the known KLF7 target Cdkn1a (p21(Cip1/Waf1)) was not induced by KLF7, and loss of CDKN1A does not rescue the repopulating defect. These results suggest that KLF7 is not required for normal hematopoietic stem and progenitor function, but increased expression, as seen in a subset of lymphoid leukemia, inhibits myeloid cell proliferation and promotes early thymocyte survival.

A Phase I Trial of Sunitinib and Rapamycin in Patients with Advanced Non-Small Cell Lung Cancer

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.

Abstract 693: Inhibitory effect of the CDK4/6 inhibitor, PD 0332991, is enhanced by mTOR inhibition in Non-Small Cell Lung Cancer (NSCLC).

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

DNA Methylation and Histone Deacetylation: Interplay and Combined Therapy in Cancer

In mammalian cells, DNA can be modified by methylation of cytosine residues in CpG dinucleotides, and the N-terminal tails of histone proteins are subject to a wide range of different modifications, including acetylation, methylation, phosphorylation and ubiquitylation. All of these chemical changes have a substantial influence on chromatin structure and gene expression. These epigenetic modification patterns can be regarded as heritable marks over many cell generations. Importantly, patterns and levels of DNA methylation and histone acetylation/deacetylation are profoundly altered in human cancers. Inhibitors of DNA methytransferases (DNMTs) and histone deacetylases (HDACs) have been shown to inhibit tumor growth by reactivating epigenetically silenced tumor suppressor genes. Although DNA methylation and histone deacetylation are carried out by different chemical reactions and require different sets of enzymes, it seems that there is a biological relationship between the two systems in modulating gene repression programming. Accumulating evidence also suggests that this epigenetic cross-talk may be involved in gene transcription and aberrant gene silencing in tumors. Thus, combined therapy with both DNMT and HDAC inhibitors can be a promising approach for cancer treatment.

Design and rational for the precision medicine guided treatment for cancer pain pragmatic clinical trial

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.

Stereotactic Ablative Body Radiotherapy for Primary Non-Small-Cell Lung Cancer: Achieving Local Control with a Lower Biologically Effective Dose

Abstract 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.