Macrophage Inhibitory Cytokine-1 Research Articles

Hyperinsulinemia acutely increases serum macrophage inhibitory cytokine‐1 concentration in anorexia nervosa and obesity

Macrophage inhibitory cytokine-1 (MIC-1) plays a role in the regulation of cellular responses to stress signals and inflammation. MIC-1 has also been implicated in mediation of tumour-induced anorexia and weight loss. Increased serum concentrations of MIC-1 were found in patients with anorexia nervosa (AN), obesity and type 2 diabetes. To estimate serum MIC-1 concentration in women with AN and obese women, its regulation by hyperinsulinemia and relationship with insulin sensitivity. We examined 20 women with AN, 28 healthy normal-weight female controls and 28 obese women. Serum MIC-1 concentration was measured in the fasting state and after 2-h euglycemic hyperinsulinemic clamp. At baseline, serum MIC-1 was higher in AN in comparison with other groups (normal-weight, P = 0·018; obese, P = 0·01). Hyperinsulinemia resulted in a significant increase in serum MIC-1 concentration in the entire study population (P < 0·001) and in AN (P < 0·001), normal-weight (P = 0·002) and obese (P < 0·001) groups analysed separately. Postclamp serum MIC-1 was still higher in AN women in comparison with other groups (normal-weight, P = 0·012; obese, P = 0·023). When normal-weight and obese women were analysed together, with the exclusion of AN group, an inverse correlation between insulin sensitivity and the change in serum MIC-1 during the clamp was observed (r = -0·27, P = 0·042). Hyperinsulinemia resulted in a significant increase in serum MIC-1 in different states of adiposity. Increased serum MIC-1 in AN women might be an additional factor responsible for weight loss in this group.

Pharmacodynamic Biomarkers in the Phase 1 Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia

Abstract 1545 Background:The tumor suppressor p53 plays a critical role in protecting cells from malignant transformation by inducing target gene transcription, cell cycle arrest, and apoptosis. MDM2 is the primary regulator of p53, targeting p53 for proteosomal degradation. The small molecule antagonist RG7112, which activates p53 by antagonism of the p53-MDM2 interaction, is currently in clinical trials. Clinical data from the Phase 1 program in leukemias has previously been presented (Andreeff et al., ASH 2010). Here we present pharmacodynamic biomarker data from the ongoing leukemia clinical trial. Methods:Patients with relapsed/refractory leukemia were treated with escalating doses of study drug in two separate strata: A- AML, ALL, CML-BC; and B - CLL and S-CLL. Peripheral blood was collected for biomarkers pre-treatment and at multiple time points during the first 48 hours and on day 10. Bone marrow was collected pre-treatment, and on day 10 and 28. Specimens of blood and marrow were subjected to RBC lysis and MACS sorting (CD19, 33 or 34 Ab). After purification, the leukemic cells were divided for preparation of RNA, DNA and protein, or fresh frozen. The validated p53 transcriptional target and secreted protein, MIC-1 (macrophage inhibitory cytokine-1), was analyzed as a pharmacodynamic marker of p53 activation. Flow cytometric analysis was performed for assessment of apoptosis (CMxRos, AnnexinV) and differentiation, (CD34, CD19). Protein immunoblots for MDM2 and p53, and real-time PCR, after reverse transcription, for quantitation of 24 customized p53 targets by TaqMan low density arrays (TLDA) (Applied Biosystems) were undertaken. AmpliChip analysis of p53 mutational status was also performed. The AmpliChip p53 test detects single nucleotide substitutions and deletions in the entire coding region and splice sites of p53 (exons 2–11) by comparative analysis of probe hybridization patterns between sample and wild-type reference DNA. Results:To date, 68 patients (42 AML, 5 ALL, 1 CML-BC, 19 CLL/sCLL, 1 T-PLL) have been treated with RG7112, with doses ranging from 20 – 2430 (1215 mg BID) mg/m2/day x 10 days. P53 mutational status has been completed in 29 Arm A and 15 Arm B patients. Preliminary results reveal that 4/29 AML patients had mutations (3 MISSENSE, 1 SPLICE). 6 of 15 patients with CLL/SLL had mutations, including a patient with a 2bp deletion in exon 6, confirmed by deep sequencing, who has stable disease on therapy for 2 years. An increase in serum MIC-1 was seen at RG7112 concentrations of >2 μg/mL and was highly correlated (R2=0.4312) with exposure to RG7112 (AUC24h) at steady state. Consistent changes in p53 target genes were observed for PUMA, p21 and FDXR (ferrodoxin reductase) as early as 6 hours after the first dose; levels were found to be significantly (p<0.05) increased 6 hours after the last dose (day 10). Increases in PUMA and p21 in AML/ALL patient samples were greater in those patients whose blast percentages decreased during Cycle 1 (n=19). Linear regression analysis revealed a dose-dependent increase of p21 (r=0.65, p=0.01). In CLL patients, the same 3 genes (PUMA, p21 and FDXR) were significantly induced during treatment with RG7112. Apoptosis induction was documented by decreased mitochondrial membrane potential (CMxRos) and increased AnnexinV binding and immunoblotting confirmed PUMA and p53 induction at the protein level in selected cases. Conclusions:We report evidence of concentration-related pharmacodynamic biomarker activity on the p53 pathway in blood and marrow specimens from patients with leukemia receiving the MDM2 antagonist, RG7112. The trial is ongoing with additional dose escalation. Further clinical and biomarker analyses are in progress and will be reported. We provide proof of mechanism for MDM2 antagonism by demonstrating p53 stabilization, activation of p53 targets and the p53 pathway in leukemic cells from patients treated with RG7112. Disclosures:Andreeff:Roche: Research Funding. Younes:Genentech: Honoraria, Research Funding. Wu:Roche: Employment. Jukofsky:Roche: Employment. Vassilev:Roche: Employment. Zhi:Roche: Employment. Geho:Roche: Employment. Nichols:Hoffmann-LaRoche: Employment.

MIC-1 (a multifunctional modulator of dendritic cell phenotype and function) is produced by decidual stromal cells and trophoblasts

Macrophage inhibitory cytokine-1 (MIC-1) is a multifunctional cytokine produced in high amounts by placental tissue. Inhibiting trophoblast invasion and suppressing inflammation through inhibition of macrophage activation, MIC-1 is thought to provide pleiotropic functions in the establishment and maintenance of pregnancy. So far, little is known about the decidual cell subsets producing MIC-1 and the effect of this cytokine on dendritic cells (DCs), which are known to play a distinct role in the development of pro-fetal tolerance in pregnancy. To identify the decidual cell types expressing and secreting MIC-1, immunohistochemical staining, PCR experiments, western blot analysis and ELISAs were performed. Immature DCs (iDCs) were generated from peripheral blood-derived monocytes and differentiated in the presence of MIC-1 or dexamethasone (Dex) for control. Migratory and proliferative activity of DCs after MIC-1 exposure was investigated by migration and proliferation assay. Cytokine secretion after MIC-1 exposure was tested in isolated uNK cells, isolated CD14+ monocytes, monocyte-derived iDCs and mature DCs. Subsequently, the phenotype of DCs was studied using FACS analysis. To test the T-cell stimulatory capacity of pre-incubated DCs, mixed lymphocyte reaction was applied. Finally, the expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) after the exposure of MIC-1 to maturing DCs was analysed by western blot. Immunohistochemical staining, PCR and western blot experiments demonstrated that MIC-1 is mainly expressed by trophoblast cells and decidual stromal cells. Analysis of the MIC-1 secretion of decidual cell types by ELISA again characterized trophoblast and stromal cells as main producers. The migratory activity of iDCs was significantly induced by MIC-1. No changes in proliferative activity of DCs were observed after MIC-1 pre-incubation. The secretion of pro- or anti-inflammatory cytokines was not affected significantly by MIC-1. Studying the phenotype of DCs after MIC-1 exposure by FACS analysis, we observed that MIC-1 suppresses the expression of typical maturation molecules such as CD25 and CD83 as well as of CD86 during cytokine-induced DC maturation similar to Dex. In addition, T-cell stimulatory capacity of DCs was significantly reduced after MIC-1 exposure. MIC-1 was also able to increase slightly the expression of IDO (a key immunomodulatory enzyme promoting periphereal tolerance) in maturing DCs. We have identified MIC-1 as a novel factor (secreted by decidual cells in early pregnancy) that could promote the increase of a tolerogenic subtype of DC in decidua.

A Phase I First-in-Human Pharmacokinetic and Pharmacodynamic Study of Serdemetan in Patients with Advanced Solid Tumors

Originally isolated on the basis of its ability to induce p53, serdemetan showed potent activity in various preclinical models, inducing S-phase arrest and apoptosis in TP53 wild-type and mutant tumors. This study evaluated the safety and tolerability of serdemetan, determined the pharmacokinetic and pharmacodynamic profiles, and identified a recommended phase II dose. Patients (71) with refractory solid tumors were allocated to dose-escalating cohorts (3+3 patients each) and received oral serdemetan once daily in 21-day cycles to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLT). Plasma was collected for pharmacokinetic analyses. Paired baseline and on-treatment skin and tumor biopsies were done; blood samples were collected for pharmacodynamic analyses, including p53 and macrophage inhibitory cytokine-1 induction. The MTD of serdemetan was determined to be 350 mg once daily. During this study, grade 3 QTc prolongation was the most common DLT and nausea (66.2%) was the most frequent treatment-emergent adverse event. Serdemetan was rapidly absorbed after oral administration and exhibited dose-proportional pharmacokinetics. At steady state, mean maximum plasma concentration (C(max)) was 2,330 ng/mL and mean area under plasma concentration curve (AUC(0-24h)) was 43.0 μg.h/mL, with serdemetan 300 mg/d. There was a dose- and exposure-dependent p53 induction. One patient with breast cancer showed a partial response; 22 (38.6%) patients had stable disease. Serdemetan treatment was associated with p53 induction in both tumor and surrogate tissue pharmacodynamic studies and modest clinical activity. Although serdemetan was well tolerated with dose-proportional pharmacokinetics, exposure-related QTc liability was observed.

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) and mortality in end-stage renal disease

Elevated macrophage inhibitory cytokine-1 (MIC-1/GDF15) levels in serum mediate anorexia and weight loss in some cancer patients and similarly elevated levels occur in chronic kidney disease (CKD). Serum MIC-1/GDF15 is also elevated in chronic inflammatory diseases and predicts atherosclerotic events independently of traditional risk factors. The relationship between chronic inflammation, decreasing body mass index (BMI) and increased mortality in CKD is not well understood and is being actively investigated. MIC-1/GDF15 may link these features of CKD. Cohorts of incident dialysis patients from Sweden (n = 98) and prevalent hemodialysis patients from the USA (n = 381) had serum MIC-1/GDF15, C-reactive protein (CRP) levels and BMI measured at study entry. Additional surrogate markers of nutritional adequacy, body composition and inflammation were assessed in Swedish patients. Patients were followed for all-cause mortality. In the Swedish cohort, serum MIC-1/GDF15 was associated with decreasing BMI, measures of nutrition and markers of oxidative stress and inflammation. Additionally, high serum MIC-1/GDF15 levels identified patients with evidence of protein-energy wasting who died in the first 3 years of dialysis. The ability of serum MIC-1/GDF15 to predict mortality in the first 3 years of dialysis was confirmed in the USA cohort. In both cohorts, serum MIC-1/GDF15 level was an independent marker of mortality when adjusted for age, CRP, BMI, history of diabetes mellitus and/or cardiovascular disease and glomerular filtration rate or length of time on dialysis at study entry. MIC-1/GDF15 is a novel independent serum marker of mortality in CKD capable of significantly improving the mortality prediction of other established markers. MIC-1/GDF15 may mediate protein-energy wasting in CKD and represent a novel therapeutic target for this fatal complication.

Diagnostic Validity of Serum Macrophage Inhibitor Cytokine and Tissue Polypeptide-Specific Antigen in Pancreatobiliary Diseases

Background and Aim: Macrophage inhibitory cytokine (MIC-1) and tissue polypeptide-specific antigen (TPS) are novel markers for several inflammatory and malignant disorders, and there are no sufficient data about the utility of these antigens as serum tumor markers. We aimed at measuring the serum levels of MIC-1 and TPS in patients with benign and malignant pancreatobiliary diseases and at determining their diagnostic efficacy. Patients and Methods: Sera collected from patients with pancreatic adenocarcinomas (56 cases), periampullary carcinomas otherthan pancreatic carcinomas (15 cases), benign pancreatic diseases (31 cases), benign biliary diseases (15 cases) and healthy volunteers (33 cases) were analyzed for MIC-1 and TPS and the results were compared with CA19-9. Results: Serum MIC-1 levels increased more significantly in patients with pancreatic carcinomas than in patients with benign pancreatobiliary diseases and healthy controls (p < 0.05). MIC-1 has a similar sensitivity (81%) but a lower specificity (73 vs. 97%) than CA 19-9 in patients with pancreatic carcinomas. Serum TPS was comparable among patients with malignant and benign pancreatobiliary diseases, and healthy controls. Conclusion: MIC-1 is a valuable tumor marker for the diagnosis of pancreatic cancer. It has a good correlation with CA 19-9. TPS has no diagnostic importance to differentiate pancreatobiliary diseases.

The TGF-β superfamily cytokine, MIC-1/GDF15: A pleotrophic cytokine with roles in inflammation, cancer and metabolism

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) is associated with cardiovascular disease, inflammation, body weight regulation and cancer. Its serum levels facilitate the diagnosis and prognosis of cancer and vascular disease. Furthermore, its serum levels are a powerful predictor of all-cause mortality, suggesting a fundamental role in biological processes associated with ageing. In cancer, the data available suggest that MIC-1/GDF15 is antitumorigenic, but this may not always be the case as disease progresses. Cancer promoting effects of MIC-1/GDF15 may be due, in part, to effects on antitumour immunity. This is suggested by the anti-inflammatory and immunosuppressive properties of MIC-1/GDF15 in animal models of atherosclerosis and rheumatoid arthritis. Furthermore, in late-stage cancer, large amounts of MIC-1/GDF15 in the circulation suppress appetite and mediate cancer anorexia/cachexia, which can be reversed by monoclonal antibodies in animals. Available data suggest MIC-1/GDF15 may be an important molecule mediating the interplay between cancer, obesity and chronic inflammation.

Effect of the Phytotherapeutic Agent Eviprostat on Inflammatory Changes and Cytokine Production in a Rat Model of Nonbacterial Prostatitis

To examine the effect of the phytotherapeutic agent Eviprostat on the stromal-to-epithelial (S/E) ratio, level of macrophage infiltration, expression of the macrophage inhibitory cytokine-1 (Mic1) gene, and tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8) concentrations in prostate tissues in a rat model of nonbacterial prostatitis (NBP). Ten-month old Wistar rats were divided into 4 groups of 10: (1) NBP non-mixed feed (prostatitis control group); (2) NBP Eviprostat (0.1%) mixed feed (prostatitis Eviprostat group); (3) non-NBP non-mixed feed (nonprostatitis control group); and (4) non-NBP Eviprostat mixed-feed (nonprostatitis Eviprostat group). NBP was induced by castration followed by daily subcutaneous injection of 17β-estradiol for 30 days. Ventral prostate lobes were histopathologically examined with Masson's trichrome staining or immunostaining with antimacrophage antibody. Mic1 mRNA levels were quantified by real-time reverse transcriptase polymerase chain reaction. Tissue concentrations of TNF-α and IL-8 were determined by enzyme-linked immunosorbent assay. Stroma was the most abundant in prostatitis control rats. The mean S/E ratio in prostatitis Eviprostat rats was significantly lower than in prostatitis control rats (P < .0001). The high levels of macrophage infiltration found in prostatitis control rats were significantly reduced in prostatitis Eviprostat rats (P < .0001). The up-regulation of the Mic1 gene observed in prostatitis control rat prostates was significantly suppressed in prostatitis Eviprostat rats (P < .0001). A marked suppression of TNF-α and IL-8 secretion was also observed in prostatitis Eviprostat rats (P < .05). Eviprostat significantly suppressed the S/E ratio, level of macrophage infiltration, Mic1 gene expression, and proinflammatory cytokines/chemokines in the prostate in a rat NBP model.

Neoadjuvant MDM2 antagonist RG7112 for well-differentiated and dedifferentiated liposarcomas (WD/DD LPS): A pharmacodynamic (PD) biomarker study.

10007b Background: WD/DD LPS are a prominent subset (15%) of sarcomas, where cytotoxic treatments have limited activity. MDM2 gene amplifications (and MDM2 overexpression) are potential molecular drivers. We report a neoadjuvant study of RG7112, a small molecule MDM2 antagonist, in chemotherapy-naive primary or relapsed LPS patients (pts) eligible for tumor resection. Methods: Pts with WD/DD LPS were treated orally at 1440 mg/m2 (Ph 1 MTD) QD for 10 days on a 28 day cycle. 3 cycles were planned, followed by complete resection. PK was collected d1 and d8 in cycle 1 (C1), with tumor biopsies collected at baseline and d8. Tumor p53 mutation (AmpliChip), MDM2 amplification (SISH) and expression (PCR), apoptosis (TUNEL) and proliferation (Ki67) were compared pre-treatment and at d8, along with serum MIC-1 (macrophage inhibitory cytokine-1), a p53 transcriptional target. Results: Between June-Dec 2010, 20 pts (12 M/8 F, med. 62y) completed pre-treatment and d8 biopsy. 12 pts had WD and 8 DD LPS. 9 were untreated, 1 metastatic. To date, 13 of 14 evaluable pts had MDM2 amplification, 2/19 had p53 mutations. 13 pts completed > 1 course (med 3); 6 received only C1. High grade adverse events included vomiting (G3-4, n=2), neutropenia (Gr 3-4 n= 3), and thrombocytopenia (Gr 3-4 n=5). Platelet nadir was highly correlated to exposure (R=0.85 and 0.86 for Cmaxss and AUCss, respectively). Increase in serum MIC-1 was also highly associated with exposure (R=0.77). Preliminary tumor analyses are available on 18/20 pts. 9/14 pts had increased TUNEL activity, 9/13 decreased Ki67 by IHC. 2 pts progressed. 7 pts have been resected with 3 patients still on treatment. Conclusions: This neoadjuvant biomarker study of RG7112, a novel MDM2 antagonist in WD/DD LPS, required sequential biopsies, was feasible, and achieved rapid completion of accrual. Preliminary data are consistent with induction of apoptosis and/or decrease in proliferation after treatment with RG7112. Treatment and assessments are ongoing. Biomarker analyses (including evidence of p53 activation by RG7112), PK clinical response data will be presented and updated with biomarker data from tumors obtained at debulking surgery.

Pharmacokinetics (PK) and pharmacodynamics (PD) of RG7112, an oral murine double minute 2 (MDM2) antagonist, in patients with leukemias and solid tumors.

3039 Background: RG7112 is a selective inhibitor of p53-MDM2 binding and frees p53 from negative control, activating the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. Results of clinical PK (plasma RG7112 concentrations) and PD (serum macrophage inhibitory cytokine-1, MIC-1, a potential indicator of p53 activation) are reported here from two phase 1 studies of RG7112 in patients (pts) with AML, ALL, CML in blast phase or refractory CLL/SCLL, and pts with advanced solid tumors and refractory lymphomas. Safety, tolerability, and clinical responses are reported separately. Methods: 49 leukemia pts and 76 solid tumor pts took RG7112 tablets once or twice daily in escalating oral doses (20 – 1920 mg/m2/day) for 10 days followed by 18 days of rest between cycles. Serial PK samples were drawn pre-dose and up to 120 hours post-dose on Days 1 and 10 and pre-dose on Day 5. MIC-1 samples were collected at the anticipated PK Cmax and up to 48-hr post- dose on Days 1 and 10. Limited confirmatory PK and PD samples were also taken in cycle 2. Results: Plasma RG7112 concentrations peaked ~ 4 hr post-dose at steady-state (SS) on Day 10 and then declined with a mean terminal elimination t1/2 ~ 1.5 days. The PK exposure (plasma Ctrough, Cmax, and AUC) was ~ dose-proportional without accumulation in the dose range studied. Inter-patient variability was ~70%; PK exposure tended to be higher in patients with solid tumors compared to those with leukemias at comparable dose levels. Serum MIC-1 levels were elevated from individual baseline starting from RG7112 doses > 320 mg/m2 and plasma concentrations > 1 µg/mL, suggesting a potential mechanistic PD relationship (p53 activation). The increase in MIC-1 (% of change from baseline) was highly associated with plasma RG7112 concentrations (R=0.60 and 0.80 in pts with leukemias and solid tumors, respectively). Conclusions: As expected from mechanism of action, RG7112 exhibited exposure-related PD response for the p53 biomarker MIC-1 within the dose range tested in pts with leukemias and solid tumors.

Pathways of chemotherapy resistance in castration-resistant prostate cancer

Chemotherapy remains the major treatment option for castration-resistant prostate cancer (CRPC) and limited cytotoxic options are available. Inherent chemotherapy resistance occurs in half of all patients and inevitably develops even in those who initially respond. Docetaxel has been the mainstay of therapy for 6 years, providing a small survival benefit at the cost of significant toxicity. Cabazitaxel is a promising second-line agent; however, it is no less toxic, whereas mitoxantrone provides only symptomatic benefit. Multiple cellular pathways involving apoptosis, inflammation, angiogenesis, signalling intermediaries, drug efflux pumps and tubulin are implicated in the development of chemoresistance. A thorough understanding of these pathways is needed to identify biomarkers that predict chemotherapy resistance with the aim to avoid unwarranted toxicities in patients who will not benefit from treatment. Until recently, the search for predictive biomarkers has been disappointing; however, the recent discovery of macrophage inhibitory cytokine 1 as a marker of chemoresistance may herald a new era of biomarker discovery in CRPC. Understanding the interface between this complex array of chemoresistance pathways rather than their study in isolation will be required to effectively predict response and target the late stages of advanced disease. The pre-clinical evidence for these resistance pathways and their progress through clinical trials as therapeutic targets is reviewed in this study.

Low AZGP1 expression predicts for recurrence in margin‐positive, localized prostate cancer

Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate at 5 years. Adjuvant radiotherapy improves biochemical progression-free and overall survival in men with positive margins, but is associated with increased toxicity. There is an urgent need to identify new prognostic markers to define the group of patients who would benefit from multimodality therapy. Nuclear β-catenin, membranous secreted frizzled-related protein 4 (sFRP4), zinc-alpha 2-glycoprotein (AZGP1), and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC. From these published studies, we identified a subset of patients with positive margins. The aim of this study was to assess the association between these four molecular markers and outcome in men with margin-positive, localized PC. We identified 186 men with positive margins from 330 men with localized PC; 53% had preoperative PSA >10 ng/ml, 72% extraprostatic extension (EPE), 24% seminal vesicles involvement (SVI), and 57% RP Gleason score ≥ 7. AZGP1 (P = 0.009), membranous sFRP4 (P = 0.03) and MIC-1 (P = 0.04) expression predicted for biochemical relapse on univariate analysis. Only absent/low AZGP1 expression (P = 0.01) was an independent predictor of recurrence in margin-positive, localized PC when modeled with preoperative PSA (P = 0.2), EPE (P = 0.2), SVI (P = 0.4), Gleason score ≥ 7 (P = 0.5) and adjuvant treatment (P = 0.4). Furthermore, there was an association between absent/low AZGP1 expression and clinical recurrence (P = 0.007). AZGP1 is a potential molecular marker for biochemical relapse in men with margin-positive, localized PC. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy.

A Multi-Center, Open-Label, Phase I Study of Single Agent RG7112, A First In Class p53-MDM2 Antagonist, In Patients with Relapsed/Refractory Acute Myeloid and Lymphoid Leukemias (AML/ALL) and Refractory Chronic Lymphocytic Leukemia/Small Cell Lymphocytic Lymphomas (CLL/SCLL)

AbstractAbstract 657 Background:Tumor suppressor p53 is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in cancer by gene mutations or defective signaling. TP53 mutations are rare in hematopoietic malignancies but activity of wild-type p53 is frequently blunted by ATM inactivation, CDKN2A (INK4A–ARF) deletion, or over-expression of MDM2. Activation of p53 by antagonism of its negative regulator MDM2 has been proposed as a novel strategy for cancer therapy. Small-molecule MDM2 antagonists, the Nutlins (Vassilev et al. Science, 2004), have been used to validate this concept preclinically in solid tumors (Tovar et al., 2006) and acute/chronic leukemias/lymphomas (Kojima et al, 2005 and 2006, Rassidakis et al). RG7112 is a member of the Nutlin family of MDM2 antagonists with identical mechanism-of-action as nutlin-3 but improved potency and pharmacological properties. Methods:Patients with relapsed/refractory leukemia who had adequate organ function were eligible. RG7112 was administered orally every day for 10 days followed by 18 days of rest. Primary objectives were to determine MTD and DLT, secondary PK, PD and clinical responses. Stratum A included AML, ALL, BC CML; stratum B included CLL and SLL. Cohorts of 3 patients at each dose level were escalated separately on the 2 strata. Dose escalation and de-escalation was based on NCI-CTCAE version 3.0 toxicity grades. Results:To date, 47 patients (27 AML, 3 ALL, 1 CML-BC, 13 CLL, 2 SLL, 1 T-PLL) have been treated with RG7112. Starting dose was 20 mg/m2/day, present dose is 810 mg/m2/day for stratum A and 1920 mg/m2/day for stratum B, with continuing escalation. Ten patients experienced moderate to severe nausea and/or vomiting, requiring anti-emetics, with 3 requiring dose modification or discontinuation. One patient had Grade 3 hyponatremia. Pharmacokinetic (PK) data demonstrate an approximate linear increase in plasma drug exposure with dose, despite PK variability. Peak concentration occurred 3–6 hours post dose with a half-life >24 hours. The p53 transcriptional target and secreted protein, MIC-1 (macrophage inhibitory cytokine-1), was evaluated as a pharmacodynamic marker of p53 activation (Yang et al., 2003). An increase in serum MIC-1 was seen at RG7112 concentrations of >1 μg/mL. Stabilization of p53 protein and induction of other p53 target genes was also observed, including CDKN1A/P21, BAX, NOXA, PUMA, FAS. Starting at 360 mg/m2, evidence of clinical activity has been observed: one patient with relapsed AML achieved complete remission (CR) that is ongoing for more than 9 months and decreased WBC and apoptosis induction were seen in CLL/SLL patients. Enrollment is ongoing and will be updated. Conclusions:We here report the first clinical trial of an MDM2 antagonist, RG7112. The molecule is well tolerated with continuing dose escalation. We provide proof-of-principle by demonstrating p53 stabilization, activation of p53 targets and the p53 pathway. One CR has been observed in AML and reductions in lymph node/spleen size and circulating leukemia cells in CLL/SLL. Future single agent and combination studies of RG7112 in leukemias are warranted. Disclosures:Andreeff:Roche: Research Funding. Padmanabhan:Roche: Research Funding. Strair:Roche: Research Funding. Kirschbaum:Roche: Research Funding. Maslak:Roche: Research Funding. Hillmen:Roche: Research Funding. Vassilev:Roche: Employment. Nichols:Roche: Employment.

Macrophage inhibitory cytokine‐1 (MIC‐1/GDF15): a new marker of all‐cause mortality

Macrophage inhibitory cytokine-1 (MIC-1/GDF15) is a member of the TGF-b superfamily, previously studied in cancer and inflammation. In addition to regulating body weight, MIC-1/GDF15 may be used to predict mortality and/or disease course in cancer, cardiovascular disease (CVD), chronic renal and heart failure, as well as pulmonary embolism. These data suggested that MIC-1/GDF15 may be a marker of all-cause mortality. To determine whether serum MIC-1/GDF15 estimation is a predictor of all-cause mortality, we examined a cohort of 876 male subjects aged 35-80 years, selected from the Swedish Population Registry, and followed them for overall mortality. Serum MIC-1/GDF15 levels were determined for all subjects from samples taken at study entry. A second (independent) cohort of 324 same-sex twins (69% female) from the Swedish Twin Registry was similarly examined. All the twins had telomere length measured and 183 had serum levels of interleukin 6 (IL-6) and C-reactive protein (CRP) available. Patients were followed for up to 14 years and had cause-specific and all-cause mortality determined. Serum MIC-1/GDF15 levels predicted mortality in the all-male cohort with an adjusted odds ratio (OR) of death of 3.38 (95%CI 1.38-8.26). This finding was validated in the twin cohort. Serum MIC-1/GDF15 remained an independent predictor of mortality when further adjusted for telomere length, IL-6 and CRP. Additionally, serum MIC-1/GDF15 levels were directly correlated with survival time independently of genetic background. Serum MIC-1/GDF15 is a novel predictor of all-cause mortality.

Growth/differentiation factor-15 is an abundant cytokine in human seminal plasma

Transforming growth factor-β cytokines have various biological effects in female reproductive tissue, including modulation of inflammatory response and induction of immune tolerance to seminal antigens in the reproductive tract. However, no studies have analyzed the presence of growth/differentiation factor-15 (GDF-15/macrophage inhibitory cytokine-1) in seminal fluid or demonstrated the quantity and form of GDF-15, its possible role or the relationship between its concentration and semen quality. The form and the concentration of GDF-15 were determined in 53 seminal plasma samples of both fertile and infertile men by ELISA and western blot. The sperm cells of three volunteers were treated with recombinant GDF-15, and cell viability and apoptosis were assessed by flow cytometry. The effect of GDF-15 on vaginal epithelial cells and peripheral blood mononuclear cells (PBMCs) was analyzed by quantitative RT-PCR. The GDF-15 concentration in seminal plasma ranged from 0.2 to 6.6 μg/ml as determined by ELISA. Western blot analysis revealed that GDF-15 is present in the active form. In vitro cultivation of sperm cells with GDF-15 did not affect their viability or rates of apoptosis; however, it did inhibit proliferation of PBMCs and induce expression of FOXP3 in CD4+CD25+ cells. To the best of our knowledge, this is the first demonstration that GDF-15 is an abundant cytokine in seminal plasma, although its concentration is not associated with semen quality or the fertility/infertility status of the donors. Moreover, our data show that GDF-15 displays immunosuppressive characteristics.

Divergent molecular mechanisms underlying the pleiotropic functions of macrophage inhibitory cytokine‐1 in cancer

Multifunctional macrophage inhibitory cytokine-1, MIC-1, is a member of the transforming growth factor-beta (TGF-beta) superfamily that plays key roles in the prenatal development and regulation of the cellular responses to stress signals and inflammation and tissue repair after acute injuries in adult life. The stringent control of the MIC-1 expression, secretion, and functions involves complex regulatory mechanisms and the interplay of other growth factor signaling networks that control the cell behavior. The deregulation of MIC-1 expression and signaling pathways has been associated with diverse human diseases and cancer progression. The MIC-1 expression levels substantially increase in cancer cells, serum, and/or cerebrospinal fluid during the progression of diverse human aggressive cancers, such as intracranial brain tumors, melanoma, and lung, gastrointestinal, pancreatic, colorectal, prostate, and breast epithelial cancers. Of clinical interest, an enhanced MIC-1 expression has been positively correlated with poor prognosis and patient survival. Secreted MIC-1 cytokine, like the TGF-beta prototypic member of the superfamily, may provide pleiotropic roles in the early and late stages of carcinogenesis. In particular, MIC-1 may contribute to the proliferation, migration, invasion, metastases, and treatment resistance of cancer cells as well as tumor-induced anorexia and weight loss in the late stages of cancer. Thus, secreted MIC-1 cytokine constitutes a new potential biomarker and therapeutic target of great clinical interest for the development of novel diagnostic and prognostic methods and/or cancer treatment against numerous metastatic, recurrent, and lethal cancers.

Expression of Macrophage Inhibitory Cytokine-1 in Benign and Malignant Prostatic Tissues: Implications for Prostate Carcinogenesis and Progression of Prostate Cancer

Expression of Macrophage Inhibitory Cytokine-1 in Benign and Malignant Prostatic Tissues: Implications for Prostate Carcinogenesis and Progression of Prostate Cancer

Macrophage Inhibitory Cytokine-1 Regulates Melanoma Vascular Development

Expression of macrophage inhibitory cytokine-1 (MIC-1), a member of the transforming growth factor-beta family, normally increases during inflammation or organ injury. MIC-1 is also expressed at higher levels in melanomas; however, its role in tumorigenesis is unknown. This report identifies a novel function for MIC-1 in cancer. MIC-1 was overexpressed in approximately 67% of advanced melanomas, accompanied by fivefold to six-fold higher levels of secreted protein in serum of melanoma patients compared with normal individuals. Constitutively active mutant (V600E)B-Raf in melanoma regulated downstream MIC-1 expression. Indeed, small-interfering RNA-mediated targeting of MIC-1 or (V600E)B-Raf reduced expression and secretion by three-fold to fivefold. This decrease in MIC-1 levels reduced melanoma tumorigenesis by approximately threefold, but did not alter cultured cell growth, suggesting a unique function other than growth control. Instead, inhibition of MIC-1 was found to mechanistically retard melanoma tumor vascular development, subsequently affecting tumor cell proliferation and apoptosis. This role in melanoma angiogenesis was confirmed by comparing MIC-1 and vascular endothelial growth factor (VEGF) function in chick chorioallantoic membrane and matrigel plug assays. Similar to VEGF in melanomas, MIC-1 stimulated directional vessel development, acting as a potent angiogenic factor. Thus, MIC-1 is secreted from melanoma cells together with VEGF to promote vascular development mediated by (V600E)B-Raf signaling.

The TGF-β Superfamily Cytokine MIC-1/GDF15: Secretory Mechanisms Facilitate Creation of Latent Stromal Stores

Macrophage inhibitory cytokine-1/growth differentiation factor 15 (MIC-1/GDF15), a divergent member of the TGF-beta superfamily is induced by a range of proinflammatory cytokines and oxidized low-density lipoprotein (oxLDL) and is highly expressed in macrophages in atherosclerotic and tumor lesions. MIC-1/GDF15, a major p53 target gene, is largely described to have anti-tumorigenic activity and more recently high MIC-1/GDF15 serum levels in late stage cancer were shown to be the major cause of cancer-associated weight loss. MIC-1/GDF15 serum levels independently predict both atherosclerotic events and severity of rheumatoid arthritis (RA), suggesting serum levels are important in modifying disease expression. Controlling serum levels is the ratio of latent unprocessed MIC-1/GDF15 stromal stores to soluble mature MIC-1/GDF15 generated by the cell. Here, we investigate MIC-1/GDF15 secretion from U937 monocytoid cells and identify novel mechanisms designed to ensure secretion of unprocessed cytokine and creation of latent stromal stores. We find that endogenous MIC-1/GDF15 is secreted as both processed and unprocessed forms. Pulse chase analysis of MIC-1/GDF15 secretion reveals that unprocessed MIC-1/GDF15 precursor is rapidly secreted, while mature MIC-1/GDF15 generated within the cell by intracellular processing is secreted much slower, possibly via an alternate secretory route. The COOH-T 47 amino acids of the propeptide are responsible for rapid secretion of MIC-1/GDF15 precursor and this effect occurs in the trans-Golgi network (TGN)/post TGN compartment. Thus, variations in MIC-1/GDF15 intracellular processing, regulating the presence or absence of propeptide, are a powerful mechanism modulating rate of MIC-1/GDF15 secretion and proMIC-1/GDF15 stromal storage, with major impact on circulating levels of mature MIC-1/GDF15.

Molecular markers that predict for recurrence in men with margin-positive localized prostate cancer.

4650 Background: Men with positive margins after radical prostatectomy (RP) for localized prostate cancer (PC) have a 40-50% biochemical relapse rate. Adjuvant radiotherapy after RP improves biochemical progression-free survival in patients with positive surgical margins, extraprostatic extension (EPE) and/or seminal vesicle involvement (SVI) but is associated with a 5-fold increase in bladder and rectal toxicity. There is an urgent need to identify new prognostic markers to better define the group of patients who would benefit from multimodality therapy. Methods: Nuclear b-catenin, membranous secreted frizzled-related protein 4 (sFRP4), AZGP1 and macrophage inhibitory cytokine-1 (MIC-1) have previously been identified as molecular markers of outcome in localized PC by our group. From these previous studies, 186 localized PC cases had positive margins. Using Kaplan-Meier and Cox Proportional Hazards analyses, we assessed the association between these four molecular markers and biochemical relapse in men with margin-positive localized PC. Results: Among the published cohort of 330 men with localized prostate cancer, we identified 186 with positive margins post RP; 53% had preoperative PSA >10 ng/ml, 72% EPE, 24% SVI and 57% RP Gleason score ≥ 7. There were 67/186 (36%) men who received adjuvant therapy (46 hormonal treatment and 21 radiotherapy). Three of the four molecular markers predicted for biochemical relapse on univariate analysis: AZGP1 (p=0.009), membranous sFRP4 (p=0.03) and MIC-1 (p=0.04). Only absent or low AZGP1 expression (HR 2.2, 95% CI 1.2-4.0, p=0.01) was an independent predictor of recurrence in margin positive localized PC when modeled with preoperative PSA (HR 1.6, 95%CI 0.8-3.1, p=0.2), EPE (HR 1.5, 95%CI 0.7-3.3, p=0.2), SVI (HR 1.4, 95%CI 0.7-3.0, p=0.4), Gleason score ≥ 7 (HR 1.3, 95%CI 0.6-2.9, p=0.5) and adjuvant treatment (HR 1.4, 95%CI 0.7-2.6, p=0.4) in a multivariate analysis. Conclusions: AZGP1 is a potential molecular marker for biochemical relapse in men with localized PC and positive margins after RP. Routine assessment of this biomarker may lead to better selection of patients who will benefit from post-RP radiotherapy and avoid morbidity in men at less risk. No significant financial relationships to disclose.