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.

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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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3102 Identifying the optimal patients and timing for allogeneic hematopoietic stem cell transplant (HSCT) is an ongoing challenge for transplant centers that requires assessment of more than just the disease status and stage. Pre-existing co-morbidities also independently impact both 100 day and 1 year non-relapse mortality (NRM) as well as long term overall survival. Additionally, the CIBMTR has also shown that Hispanic patients have an inferior survival after allogeneic HSCT, although why ethnicity is linked to a higher mortality, e.g. is it higher risk/timing of transplant, lower socioeconomic status, increased co-morbidities, etc. however, is still undetermined. A survival analysis of allogeneic HCT outcomes that includes ethnicity, co-morbidity, insurance payor, as well as traditional risk factors such as age, disease risk at transplant, and remission status would be helpful to clarify the outcome disparities found in different ethnic subgroups and more importantly lead to strategies to minimize the effect. Methods: We performed a retrospective analysis of the outcome of 363 consecutive adult allogeneic transplants at Loyola University Hospital of whom approximately 10% were Hispanics (34) from January 1, 2003 to June 30, 2010. We use a focused approach to optimize care of non-English speaking Hispanics which involves native speakers at all levels, written materials in Spanish, and an IRB approved method to permit all non-English speaking patients to enroll in all clinical trials is present. Traditional prognostic data as well as the Sorror co-morbidity index and socioeconomic and ethnicity status were determined for each patient. The surrogate we used for socioeconomic status was insurance payor (i.e. indigent + Medicaid vs. third party insurance/Medicare). Survival analysis was calculated using Kaplan Meier plots considering each patient at 100 days, 1 year, and 3 years. Results: The median age for the 363 patients was 47.1 years with 48.7% being in the High Risk CIBMTR category, and 74.1% being in remission at transplant. The median co-morbidity index was 2.0. No differences were seen in remission status or the co-morbidity index for Hispanic vs. non-Hispanic patients, while fewer Hispanic patients were in the High CIBMTR Risk group (27.3 vs. 50.9%; p = .048). Survival for Hispanics (34) vs. non-Hispanics (329) showed no difference in overall survival at three years (42.1 vs. 42.8%). Based on payors, we found no statistical difference in 100 day overall mortality after HSCT for those with Medicaid (n = 35) vs. non-Medicaid (n = 328) payors with Hispanic non-Medicaid and non-Hispanic non-Medicaid survivals at 100 days of 82.8 and 87.9%. However despite the relatively small number, both the non-Hispanic and Hispanic patients with Medicaid showed a significantly decreased survival after 100 days with 3 year survivals of 20.0 and 30.0% for Hispanic and non-Hispanic Medicaid patients vs. 51.7 and 56.7% of the Hispanic and non-Hispanic non-Medicaid groups respectively (p = .002). Conclusions: Unlike prior reports, and perhaps by using a focused approach for our non-English speaking Hispanic patients, we found no difference in survival between Hispanics and non-Hispanics in our series of 363 consecutive allografts at 100 days, 1, or 3 years, although they may have been predicted to have had a slightly better prognosis based on a better Risk Group status. We did however find that payor was an important prognostic variable for outcome after the first 100 days regardless of ethnicity. Whether this is simply due to the financial toll of such a chronic illness and therapy remains to be seen, but this suggests that closer follow-up after day 100 may be of significant benefit for this subgroup of patents which may grow due to health care reform. Disclosures: No relevant conflicts of interest to declare.

Despite many available effective agents for multiple myeloma (MM), the development of drug resistance is inevitable and novel regimens are needed. BET (bromodomain and extra-terminal) family proteins include BRD2, BRD3, BRD4, and BRDT. Bromodomain (BRD)-containing proteins are the principal readers of the acetyl lysine marks placed by histone modifying enzymes (HATs, HDACs). It has been previously shown that targeted BET inhibition leads to multiple alterations in gene expression. Preclinical data has shown that the BET inhibitor JQ1 is effective in MM, and is associated with multiple changes in gene transcription including the downregulation of c-myc. We tested CPI203, a recently characterized selective BET inhibitor (BRD4 IC50=26 nM, CBP IC50= 6000nM), in preclinical models of drug resistant multiple myeloma. Single agent CPI203 inhibits proliferation in 6 different MM cell lines including bortezomib (BTZ)- and melphalan-resistant lines. The mean IC50 across the cell lines was 1206 nM (range 142-6919 nM). Interestingly, CPI203 was more potent across all drug resistant cell lines (8226.BR, ANBL6 BR, 8226/LR5) with a mean IC50 of 216 nM (range 142-310) compared to the “drug sensitive” parent lines (RPMI 8226, ANBL6 WT, U266) that had a mean IC50 of 1801 nM (range 220-6919 nM). In particular, CPI203 was 22-fold more potent in the BTZ resistant cell line (ANBL6 BR, IC50=310 nM) than in its parent line (ANBL6 WT, IC50=6919 nM). The combination of BTZ and CPI203 was synergistic in ANBL6 WT (combination index (CI) of 0.61-0.79), ANBL6 BR (CI 0.35-0.71), as well as in the melphalan resistant line, 8226/LR5 (CI 0.57-0.93), but not in RPMI 8226 WT. Furthermore, BTZ + CPI203 treatment of a primary bone marrow sample from a patient with 17p deleted relapsed-refractory MM who recently had progressive disease on a proteasome inhibitor demonstrated synergy in a cell viability assay (CI 0.24-0.71). To assess the mechanism of cell death, an apoptosis assay was performed after treatment of ANBL6 WT and ANBL6 BR with BTZ and CPI203. As expected, single agent BTZ induced dose dependent accumulation of cells in early apoptosis, and to a lesser extent late apoptosis. At low concentrations of single agent CPI203 (40-160 nM), there was undetectable difference in apoptosis over untreated controls. However, treatment with 1600 nM CPI203 resulted in a 57% and an 89% relative increase in the percentage of apoptosis in ANBL6 WT and ANBL6 BR respectively. Treatment of ANBL6 WT cells with the combination of 1600nM CPI203 + 20nM of BTZ led to apoptosis in 94% of the cells, compared 40% apoptosis in untreated (no drug) cells (p=0.0017). Likewise in ANBL6 BR cells, 76% of the cells treated with the combination of 1600nM CPI203 + 20 nM BTZ were apoptotic vs 19% in untreated cells (p=0.0001). The combination index in ANBL6 BR was 0.7, again consistent with synergy. Studies have illustrated that the effects of proteasome inhibition converge with the induction of NOXA, a proapoptotic protein that triggers cell death through both caspase- and non-caspase-dependent pathways. NOXA has been shown to be under transcriptional regulation by c-myc. Given that small molecule BET inhibitors can reduce the expression of c-myc, we further explored the synergistic mechanism between BTZ and CPI203. Immunoblotting of ANBL6 WT and BR cell lysates treated with BTZ and CPI203 for NOXA and c-myc revealed that single agent BTZ induced the expected dose-dependent rise in NOXA, but did not appear to impact c-myc expression levels. Single agent CPI203 led to a dose-dependent decrease in c-myc, but had little effect on NOXA expression. When BTZ was combined with CPI203, there was a relative decrease in NOXA induction compared to cells treated with BTZ in the absence of CPI203. The combination did not appear to have any further impact on c-myc levels compared to the effects of CPI203 alone. While the mechanism of the synergistic relationship is not clearly understood at this time, the effectiveness of the combination is compelling and is consistent across multiple in vitro settings. In conclusion, the BET inhibitor CPI203 shows favorable activity in drug resistant MM, and is synergistic with BTZ in human myeloma cell lines as well as in primary MM cells at low nanomolar concentrations, supporting further clinical investigation of this active combination. 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. Tyner:Constellation Pharmaceuticals: Research Funding.