ALBERT

Description: Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala−mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP−mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP−mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala−mDAP–specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis.

Description: Bacterial growth and morphogenesis are intimately coupled to expansion of peptidoglycan (PG), an extensively cross-linked macromolecule that forms a protective mesh-like sacculus around the cytoplasmic membrane. Growth of the PG sacculus is a dynamic event requiring the concerted action of hydrolases that cleave the cross-links for insertion of new material and synthases that catalyze cross-link formation; however, the factors that regulate PG expansion during bacterial growth are poorly understood. Here, we show that the PG hydrolase MepS (formerly Spr), which is specific to cleavage of cross-links during PG expansion inEscherichia coli, is modulated by proteolysis. Using combined genetic, molecular, and biochemical approaches, we demonstrate that MepS is rapidly degraded by a proteolytic system comprising an outer membrane lipoprotein of unknown function, NlpI, and a periplasmic protease, Prc (or Tsp). In summary, our results indicate that the NlpI–Prc system contributes to growth and enlargement of the PG sacculus by modulating the cellular levels of the cross-link–cleaving hydrolase MepS. Overall, this study signifies the importance of PG cross-link cleavage and its regulation in bacterial cell wall biogenesis.

Description: Tumor protein D52 (TPD52), a proto-oncogene is overexpressed in a variety of epithelial carcinomas and plays an important role in cell proliferation, migration and cell death. In the present study we found that the treatment of IMR-32 neuroblastoma (NB) cells with retinoic acid (RA) stimulates an increase in expression of TPD52. TPD52 expression is detectable after 72 h, can be maintained till differentiation of NB cells suggesting that TPD52 is involved in differentiation. Here, we demonstrate that TPD52 is essential for RA to promote differentiation of NB cells. Our results show that exogenous expression of EGFP-TPD52 in IMR-32 cells resulted cell differentiation even without RA. RA by itself and with overexpression of TPD52 can increase the ability of NB cells differentiation. Interestingly, transfection of IMR-32 cells with a specific small hairpin RNA for efficient knockdown of TPD52 attenuated RA induced NB cells differentiation. Transcriptional and translational level expression of neurotropic (BDNF, NGF, Nestin) and differentiation (β III tubulin, NSE, TH) factors in NB cells with altered TPD52 expression and/or RA treatment confirmed essential function of TPD52 in cellular differentiation. Furthermore, we show that TPD52 protects cells from apoptosis and arrest cell proliferation by varying expression of p27Kip1, activation of Akt and ERK1/2 thus promoting cell differentiation. Additionally, inhibition of STAT3 activation by its specific inhibitor arrested NB cells differentiation by EGFP-TPD52 overexpression with or without RA. Taken together, our data reveal that TPD52 act through activation of JAK/STAT signaling pathway to undertake NB cells differentiation induced by RA. This article is protected by copyright. All rights reserved

Description: Human herpesvirus-8(HHV-8)-negative/idiopathic multicentric Castleman disease (iMCD) is a rare and poorly understood disorder diagnosed in ~1,000 individuals in the USA each year. It involves polyclonal lymphoproliferation, constitutional symptoms, systemic inflammation and an uncontrollable cytokine storm resulting in life-threatening multi-organ failure. Diagnosis and treatment can be difficult due to limited etiological understanding and heterogeneous presentation - clinical, laboratory, and histopathological abnormalities overlap with infectious, autoimmune and oncological diseases. iMCD symptoms and disease progression are largely believed to be driven by interleukin-6 (IL-6). However, approximately 66% of iMCD patients did not respond to anti-IL-6 therapy, siltuximab, the only FDA-approved iMCD therapy, in its phase II study (NCT01024036). Few treatment options exist for anti-IL-6 refractory patients because alternative driver cytokines and signaling pathways are not known. Herein we report the largest study to-date of iMCD serum proteomes with correlative anti-IL6 response data from 92 iMCD patients in disease flare (n=75 of which were collected as part of NCT01024036), in order to: (1) molecularly define iMCD, (2) identify predictors of response to anti-IL6 therapy, and (3) gain insights into the pathogenesis of iMCD. Proteomes of HHV8-positive MCD (n=20), Hodgkin lymphoma (n=20), rheumatoid arthritis (n=20) and healthy individuals (n=44) were also analyzed. Of the ~1,300 analytes measured using SomaLogic SOMAscan, 1,178 passed QC and were included in analyses. Each analyte was log2 transformed and capped at the 2.5th and 97.5th percentiles. Clinical and laboratory data collected at the time of sample draw were used to calculate disease activity following a modified CHAP scale: C-reactive protein, hemoglobin and albumin; missing performance status. Response to siltuximab was determined in NCT01024036. Data analysis was performed using the Medidata Rave Omics machine learning platform and R v3.4.4. Clustering of baseline proteomic data for iMCD patients identified six clusters that ranged in size from seven to 27 subjects. No associations with race, site, sex, age, or batch were found. Analytes identified among the strongest differentiators include cytokines, chemokines and inflammatory molecules. Interestingly, the largest cluster was associated with response to siltuximab (p

Description: Abstract 1365 Introduction Siltuximab (CNTO 328) is a chimeric, murine-human, monoclonal antibody that specifically binds human interleukin (IL)-6 with high affinity. C-reactive protein (CRP) can be a pharmacodynamic (PD) marker of IL-6 bioactivity, i.e., reductions in CRP suggest inhibition of systemic IL-6. A population mechanistic pharmacokinetic (PK)/PD model was developed to describe the relationship between siltuximab serum concentrations and CRP suppression in patients with B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), or Castleman's disease (CD). Simulation was used to support the dose selection in the CD registration study and future clinical studies. Methods PK/PD data were obtained from a phase 1 clinical study examining multiple dosing regimens of siltuximab administered intravenously in patients with NHL, MM, or CD. Dosing regimens included siltuximab 2.8, 5.5, or 11 mg/kg every 2 weeks; 11 mg/kg every 3 weeks; or 5.5 mg/kg every week. Serial samples to determine serum concentration of siltuximab and serial CRP samples were collected following the first dose. NONMEM 7 was used to simultaneously fit a two-compartment PK model and an inhibitory indirect-response PD model to the observed data. Simulation of 1000 replications was then used to identify siltuximab dosage regimens that would maintain CRP suppression below the lower limit of quantification (LLOQ) of 1 mg/L. Results The mechanistic PK/PD model was able to describe the serum siltuximab and CRP concentration-time profiles. Volume of distribution and systemic clearance rate constant of siltuximab were estimated at 68.42 mL/kg and 0.0584/day, respectively. The PD parameter estimates (Kin and Kout of CRP) were 5.03 mg/L/day and 0.457/day, respectively, and were similar between the three disease types in this study. IC50was estimated at 9.73 μg/mL and was also similar between disease types. For all disease types, simulations showed that siltuximab 11 mg/kg every 3 weeks or 15 mg/kg every 4 weeks after the second dose would reduce serum CRP to below the LLOQ throughout the entire treatment period. However, lower dose intensive schedules, including a dose of 5.5 mg/kg every 2 weeks, would not reduce CRP to below the LLOQ at any time point during the treatment period. Conclusion The population PK/PD modeling and simulation support using a siltuximab dose of 11 mg/kg every 3 weeks or 15 mg/kg every 4 weeks in future clinical development studies. This dosing recommendation is supported by the observed efficacy dose-response relationship in patients with CD (J Clin Oncol 2010;28:3701–8). Disclosures: Xie: Johnson & Johnson: Employment, Equity Ownership. Li:Johnson & Johnson: Employment, Equity Ownership. Kurzrock:Johnson & Johnson: Honoraria, Research Funding. van Rhee:Johnson & Johnson: Research Funding. Qin:Johnson & Johnson: Employment, Equity Ownership. Reddy:Johnson & Johnson: Employment, Equity Ownership. Qi:Johnson & Johnson: Employment, Equity Ownership. Davis:Johnson & Johnson: Employment, Equity Ownership. Zhou:Johnson & Johnson: Employment, Equity Ownership. Puchalski:Johnson & Johnson: Employment, Equity Ownership.

Description: Background Human herpes virus-8 (HHV8) negative/idiopathic multicentric Castleman disease (iMCD) is a rare hematological disorder with one FDA-approved treatment and no early indicators of patient response after treatment is commenced. Clinical onset can be sudden and severe, involving polyclonal lymphoproliferation with characteristic dysmorphic germinal centers, constitutional symptoms, systemic inflammation and life-threatening cytokine storm-driven multi-organ failure. CXCL13, a key regulator of lymph node germinal center development, was recently found to be the most elevated cytokine in iMCD flare, but the clinical significance of this finding is not yet clear. Interleukin-6 (IL-6) is the known driver of pathogenesis in a portion of patients and the target of the only FDA-approved treatment, siltuximab. In the Phase II study of siltuximab (NCT01024036), one-third of patients met response criteria, which were assessed after a minimum of 48 weeks. Early indicators of response to siltuximab are urgently needed to inform clinicians about the likelihood of patient response to therapy, adjust treatments if needed, and identify novel therapeutic targets for siltuximab non-responders. Methods Clinical data and serum samples were collected as part of NCT01024036. We measured serum protein analytes in the 52 subjects who were treated with anti-IL6 therapy, as well as the 26 patients in the control arm, at day 1, day 8, and day 64 of therapy (infusions administered every 21 days). Serum samples from 44 healthy donors were also analyzed. Of the 1,305 analytes measured using SomaLogic SOMAscan, 1,178 passed QC and were included in analyses. Each analyte was log2 transformed and capped at the 2.5th and 97.5th percentiles. Response to anti-IL6 therapy was determined by independent review in NCT01024036. Data processing was performed using the Medidata Rave Omics machine learning platform and R v3.4.4. Linear mixed effects models were used to detect whether kinetic changes in protein expression were associated with anti-IL6 response. Upon running the full model with the selected covariates, the p-values of the interaction between time point and response were used to test for differences between responders and non-responders. A separate model was fitted using each protein, and False Discovery Rates (FDR) were estimated by the Benjamini-Hochberg method with alpha 〈 0.05. Results Seven days after siltuximab was first administered (day 8), 9 proteins were significantly different between responders and non-responders: IgA, BCMA, NPS-PLA2, ART, IL-18 BPa, CD5L, b2-Microglobulin, CXCL13, and NRP1. All 9 of these proteins were significantly decreased in responders compared to non-responders. At day 64, the number of significantly different proteins increased to 121, including 8 of the 9 proteins from day 8; NPS-PLA2 did not achieve significance at day 64. This result indicates that there may be early indicators of response in serum as early as day 8. Given that CXCL13 was recently discovered as a key cytokine in iMCD, the early and significant decline of CXCL13 in responders versus non-responders was highly notable (Day 8: FDR = 0.02, Day 64: FDR = 0.005). Prior to treatment, CXCL13 was significantly higher in this cohort of iMCD patients than in a group of age-matched healthy donors (p = 8.19e-09). By day 64, CXCL13 levels in siltuximab responders decreased to levels approaching the healthy donor range but remained elevated in non-responders and placebo patients. Conclusions This analysis represents the first use of high-quality serum proteomics data to study early indicators of response to treatment in a rare hyperinflammatory, lymphoproliferative disorder. The decline in CXCL13 levels in responders and continued elevation in non-responders suggests that CXCL13 is downstream of IL-6 in responders and independent of IL-6 signaling in non-responders. CXCL13, along with several other proteins that demonstrated significant decline by day 8 including IgA and beta2-microglobulin, can be routinely measured and could serve as a panel that indicates the likelihood of response soon after commencing therapy, if validated in a separate cohort. These proteins could also provide a more continuous scale of response than traditional outcome measures. Given that iMCD may have a sudden and severe onset, early indicators of response to anti-IL6 therapy are critical for timely treatment administration. Figure Disclosures Katz: Medidata Solutions: Employment. Nabel:Thermo Fisher: Patents & Royalties: royalty income. Diamond:Brigham and Women's Hospital and Harvard Medical School's Department of Medicine, Division of Genetics, Genomes2People Translational Genomics Research Program: Consultancy. Karvir:Medidata Solutions: Employment. Zhang:Medidata Solutions: Employment. Reddy:Janssen Pharmaceuticals: Employment, Equity Ownership. Guilfoyle:Janssen Pharmaceuticals: Employment, Equity Ownership. Tendler:Janssen Pharmaceuticals: Employment, Equity Ownership. van Rhee:Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy; Adicet Bio: Consultancy; Kite Pharma: Consultancy; Karyopharm Therapeutics: Consultancy. Beineke:Medidata Solutions: Employment. Oromendia:Medidata Solutions: Employment, Equity Ownership. Fajgenbaum:Janssen Pharmaceuticals: Research Funding.

Description: Key Points Adding siltuximab to VMP did not improve CR, progression-free survival, or overall survival but improved very good partial response in MM. This suggests that the association of less than CR with long-term outcomes and the role of IL-6 in MM should be reassessed.

Description: Abstract 4045 Poster Board III-980 Introduction CNTO328 is a chimeric monoclonal antibody against the inflammatory cytokine, IL-6, which is currently being studied in hematologic and solid malignancies. IL-6 is reported to play a key role in the etiology and symptoms of Anemia of Cancer (AOC). Increased IL-6 during cancer associated inflammation up-regulates the hepatic production of hepcidin, which is the iron-regulatory hormone that may be responsible for most of the features of this disorder. CNTO328 treatment has previously been shown to be associated with Hb increases in Castleman's Disease (a disorder caused by deregulated IL-6 production) and Renal Cell Carcinoma (RCC). We retrospectively undertook this assessment in the RCC patients, to assess whether there was an associated decrease in hepcidin. Patients and Methods Serum Hb, IL-6 and CRP (a surrogate for IL-6 activity) levels were prospectively studied in RCC patients selected from two 6 mg/kg CNTO328 treatment cohorts (IV Q2W or Q3W) in a phase 1/2 study. Free and total IL-6 could not be measured due to interference of the drug with assay performance which prevents accurate measurement. Serum hepcidin levels were retrospectively measured using a hepcidin C-ELISA. The change from baseline in Hb, hepcidin and CRP levels was calculated at multiple time points. The association between Hb response (defined as max Hb increase of ≥1 g/dL) and change in hepcidin and CRP levels was evaluated by Pearson Correlation Coefficient (r). Results 38 RCC patients with a median baseline Hb of 13.2 g/dL (10.1-17.1) were studied. All patients had normal renal function throughout the study. Two patients were excluded from analysis because of blood transfusions at baseline. None of the patients studied received ESAs or blood transfusions during screening or treatment. Treatment with CNTO328 resulted in an Hb increase during study in 35 (92%) of the 38 patients starting on Day 8, with 25 (66%) achieving a max Hb increase of 31 g/dL (median 2.0; range 1.0-3.5). Hb responses were not due to tumor response, were independent of dosing schedule (Q2W vs Q3W) and were even seen in 6 (86%) out of 7 patients with a baseline Hb