Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.
PurposeDespite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma.Experimental DesignA panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells.ResultsThe overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events.ConclusionOur preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma.
Background. Chemotherapy resistance is a major barrier to improving outcomes in relapsed and refractory MM (RRMM). Inhibition of mTOR signaling induces cell-cycle arrest, apoptosis and sensitizes MM cells to dexamethasone (dex) and alkylators. Autophagy is a lysosome-dependent degradative pathway activated in malignant cells that allows cells to survive the intracellular metabolic crisis induced by chemotherapy and signaling inhibitors. Preclinical evidence suggests that mTOR signaling and activation of autophagy are inter-related mechanisms of chemotherapy resistance. We therefore conducted a phase I trial adding the mTOR inhibitor, rapamycin (rapa) and the autophagy inhibitor, hydroxychloroquine (hcq), to a conventional chemotherapy backbone, with the primary objective of showing that the combination is well tolerated and a secondary objective of evaluating in vivo whether the regimen effectively inhibits autophagy and mTOR signaling in tumor cells. Methods. Patients with RRMM who had received > 1 prior line of therapy including bortezomib and lenalidomide were enrolled in a standard 3 + 3 dose escalation study with hcq dose levels ranging from 400 to 1200 mg. Continuous infusion cyclophosphamide (cy) 300mg/m2 /day and dex 40mg by mouth were given on days 1-4 of a 28 day cycle. Rapa 12mg loading (on day -2) and 4mg daily doses by mouth were given on days -1 to 4 and hcq was started on cycle 1 day 5 and continued daily by mouth thereafter. Pegfilgrastim 6mg was given on day 6. Dose- limiting toxicity (DLT) was defined as grade >3 adverse events as per CTCAE version 4.0 occurring during the first 2 cycles with exclusion of expected toxicities with cy. Bone marrow aspiration (CD 138 magnetic bead selected plasma cells) and biopsies were obtained at 3 timepoints: baseline, cycle 1 and 2 day 5 for pharmacodynamic assessments including: electron microscopy to evaluate autophagic vesicles, immunoblotting for LC3 II, I, phosphorylation of S6, AKT and eIF4E. Hcq and rapa trough levels were obtained from peripheral whole blood. Results.We enrolled 15 patients, between 1/2013 and 6/2014, of whom 12 completed at least 2 cycles and were evaluable for response. Toxicity evaluation included all 15 patients. Patients had received 3-10 (median 6.5) prior lines of therapy. The median number of cycles completed was 4.5 (range 1-12). 14 patients had received prior melphalan autologous stem cell transplant and 4, additional alkylator based regimens. Reasons for study discontinuation were disease progression (PD) 5, lack of response 6, treatment toxicity 2, and 2 patients remain on study. Two DLTs occurred at the 4th dose level: grade III diarrhea related to hcq and in another patient, grade IV thrombocytopenia that did not resolve possibly related to cy, hcq or rapa. The maximum tolerated dose (MTD) is hcq 800mg and this cohort has been expanded to include 3 more patients. Other significant adverse events included grade 4 thrombocytopenia 4, neutropenia 1, lymphopenia 5, grade 3 catheter associated MRSA bacteremia 2, diarrhea 2, constipation 2, abdominal discomfort 1, prolonged QTc 1. Three patients came off study after 1 cycle due to PD, toxicity and patient choice. In the remaining 12, anti-myeloma activity was seen in 9 patients: 3 partial (PR) and 6 minor (MR) responses. Stable disease (SD) was seen in 2 subjects while one had PD. Time to maximum and duration of response was 2 and 3-6 months respectively for patients achieving PR. For those achieving SD/MR, time to next treatment was 1-7 months (median 4) and not yet reached in 1. Median hcq trough levels in cohorts 1-4 were 466, 838, 1486 and 1635 ng/ml respectively (range 363-2130). Rapa trough levels were 3.2-17.8 ng/mL. Autophagic vesicle counts over time were noted to increase in responders and LC3 II:I increased in most patients on higher dose cohorts suggestive of inhibition of autophagy. Changes in signaling pathways were appreciated and results will be presented. Conclusions. The addition of mTOR and autophagy inhibition to a backbone of cy/dex yields a tolerable regimen with durable responses in heavily pretreated patients. Hcq at the MTD results in whole blood concentrations known to inhibit autophagy. In vivo assessment of autophagy and mTOR pathways is feasible and correlation with clinical response will be possible in larger studies. A randomized phase 2 study is needed to determine the synergistic properties of dual mTOR and autophagy inhibition vs chemotherapy alone. Disclosures Scott: Millenium: Membership on an entity's Board of Directors or advisory committees, Research Funding; Onyx Amgen: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Hydroxychloroquine is FDA approved for treatment of malaria and rheumatologic diseases. Rapamycicn is FDA approved for solid organ transplant prevention of rejection.
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