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Introduction: Prior to the introduction of tyrosine kinase inhibitors (TKIs), the presence of BCR-ABL1 conferred a poor prognosis in patients with acute lymphoblastic leukemia (ALL). We published in 2017 in Br J Haematology our analysis comparing the survival of Ph-Positive (Ph+) and Ph-negative ALL during the period when TKIs were universally available in the United States for Ph+ ALL using a Surveillance, Epidemiology, and End Results (SEER) Database analysis. Despite using TKIs, we have continued to remain reliant on cytotoxic chemotherapy regimens and allogeneic hematopoietic stem cell transplant (allo-HSCT) to achieve the best long-term outcomes. However, with the introduction of more potent TKIs and other novel agents, as well as better methods for monitoring minimal/measurable residual disease (MRD) the best approach is yet to be determined. In this study we present data from our institution with incorporation of TKIs in our modified USC ALL pediatric-based regimen without pegaspargase (PEG) (Table 1). Methods: This retrospective, single institution chart review included adults aged >18 with newly diagnosed Ph+ ALL between 2016 and 2020. Primary objectives were Overall survival (OS) and event-free survival (EFS) at 3 years for Ph+ ALL patients and secondary objectives were rates of complete remission/complete remission with incomplete recovery (CR/CRi), minimal residual disease (MRD) by flow cytometry and presence of BCR-ABL1 fusion transcript by real time polymerase chain reaction. Descriptive statistics of patients were reported and evaluated using Fisher's exact test. OS and EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant. RESULTS: 26 Ph+ ALL patients were identified. Median age at diagnosis was 42.5 years, with 42.3% males and 57.7% females. Median BMI was 31.1 kg/m 2, 42.3% had hepatic steatosis and 34.6% had CNS disease pre-induction. After induction 1, 91.3% of patients achieved CR/CRi, 8.7% were refractory, and 64.3% were MRD flow negative with 24% of patients with undetectable BCR-ABL1. After induction 2, 94.1% had achieved CR/CRi, 64.3% were MRD flow negative with 44.4% of patients with undetectable BCR-ABL1. After consolidation I, 78.6% were MRD flow negative. 50% of patients had received blinatumomab for MRD flow positivity early in the course and 34.6% underwent allo-HSCT. Of note, 65.4% of patients received Dasatinib only and 30.8% received at least 2 TKIs. Overall, 11.5% had known relapse, 12.5% died. 3-year OS was 83.3% and 3-year EFS was 86.6% (Table 2). When survival was stratified by transplant status, 3-year OS with allo-HSCT was 100% versus 70% without allo-HSCT (p=0.15) and 3-year EFS with allo-HSCT was 100% compared to 77.9% without allo-HSCT (Table 3). CONCLUSIONS: The use of the modified USC ALL regimen without PEG for the treatment of newly diagnosed Ph+ ALL combined with TKI at our institution led to an excellent 3-year OS (83.3%) and 3-year EFS (86.6%). All patients received TKI, half of the patients received blinatumomab and at least one third received allo-HSCT which likely led to higher OS even without PEG. We also observed a trend towards improved OS in recipients of allo-HSCT compared to patients who did not receive allo-HSCT (100% vs. 70%, p=0.15) although statistically not significant, it highlights the role of allo-HSCT in the management of Ph+ ALL patients. Figure 1 Figure 1. Disclosures Chaudhary: TCR2: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pancella: Consultancy; Oncotartis: Consultancy; Athelas: Consultancy, Current holder of stock options in a privately-held company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy ; Allogene: Current equity holder in publicly-traded company. Douer: Adaptive: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Speakers Bureau; Jazz: Consultancy; Servier: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Yaghmour: Agios: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau.

Background: Haploidentical hematopoietic cell transplantation (haplo-HCT) has emerged as a popular alternative to traditional HLA-matched hematopoietic cell transplant. The major advancement of haplo-HCT has increased donor availability to many patients in need, therefore investigating the factors that may affect outcomes is necessary to improve overall survival and reduce transplant-related mortality. Data regarding optimal dose of CD34 cells, CD3 cells and CD34/CD3 ratio used during haplo-HCT to ensure favorable outcomes with post-transplant cyclophosphamide (PTCy) is lacking. Previously we have reported improved outcomes using CD34 cells limited to 7x10^6 cells/kg or less. In this report we are presenting an updated analysis including outcomes related to CD34/CD3 ratio used during haplo-HCT. Methods: We retrospectively analyzed adult patients at USC Norris Cancer Hospital who received haplo-HCT from 2014 to 2020. The primary endpoint assessed was 1-year GVHD-free/relapse-free survival (GRFS). Secondary endpoints included overall survival (OS), 1-year transplant related mortality (TRM) and incidence of acute and chronic GVHD. Kaplan Meier survival curves and log-rank tests were used to evaluate 1-year GRFS and OS with CD34 cell dose ≥ 7x10^6 cells/kg and < 7x10^6 cells/kg; CD3 cell dose < 2.5x10^8 cells/kg and ≥ 2.5x10^8 cells/kg; and CD34/CD3 ratio 3-5 and >5. Results: 103 adult haplo-HCT recipients were reviewed with 55.3% male and 44.7% female. The age range was 21-71 years old (median = 51), and a majority of patients were Hispanic (62%). The most common underlying hematologic disorders included AML (40.8%), ALL (34.1%), and MDS/MPN (13.6 %). 41.7% patients with leukemia were in CR1, 55% were CR2/CR3 and 78% were MRD negative by flow cytometry prior to transplant. 73% received myeloablative conditioning and 83.5% received peripheral blood stem cells. Median CD34 dose, CD3 dose and CD34/CD3 ratio were 6.1 x10^6cells/kg, 2.27 x10^8 cells/kg and 3.2 respectively. Median time to recovery of neutrophil, platelets, and lymphocyte was 17, 24, and 124 days respectively. Incidence of 1-year GRFS was 43.7%. 1-year TRM was 13.6% and rate of aGVHD and cGVHD was 42.7% (n = 44) and 35.9% (n = 37) respectively. There was no difference in 1-year GRFS and OS when multiple dose levels of CD34 cells and CD3 cells were compared. Although when dichotomized, a CD34 cell dose of ≥ 7x10^6 cells/kg compared to < 7x10^6 cells/kg showed higher dose had significant improvement in 1- year overall survival (p-value=0.01) but no statistical difference in 1 year GRFS (p value=0.24). CD3 cell dose < 2.5 x 10^8 cells/kg trended towards worse 1-Year GRFS compared to ≥ 2.5 x 10^8 cells/kg (43.9% compared to 58.6%; p-value 0.075), similarly CD3 dose of < 2.5x10^8 cells/kg showed a lower 1-year OS compared to > 2.5x10^8 cells/kg but was not statistically significant (78.7% compared to 89.4%; p-value 0.092). 1-year GRFS and OS did not show statistical difference at CD34:CD3 ratios of < 2, 2-3, 3-5 and > 5, although on cox regression analysis the HR for 1-year GRFS was 2.92 (95% CI 1.21-7.05) in patients with CD34:CD3 > 5 as compared to reference of < 2 (p-value 0.017). Multivariate regression also showed CD3 cell doses of 2-3x10^8 cells/kg and >3 x 10^8 cells/kg were associated with worse 1-year GRFS with HR of 0.43 and 0.40 and p-value of 0.041 and 0.048 respectively when compared to reference of < 1x10^8 CD3 cells/kg. Discussion: Our results demonstrate 43.7% survived 1 year compared with reports of 24-35%. The OS was significantly better in the CD34 dose > 7x10^6 cells/kg. CD3 2x10^8 cells/kg was associated with inferior 1-year GRFS. Conversely, higher CD34/CD3 ratio >5 was associated with increased 1-year GRFS. Thus, our findings indicate that along with improvement in OS by using >7x10^6 CD34 cells/kg, lower CD 3 cell dose 5 can improve 1-year GRFS in patients receiving haplo-HCT. Figure 1 Figure 1. Disclosures Chaudhary: Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy ; Athelas: Consultancy, Current holder of stock options in a privately-held company; Oncotartis: Consultancy; Pancella: Consultancy; Moderna: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company. Yaghmour: Jazz: Consultancy, Honoraria; Astellas: Consultancy; Takeda: Consultancy; Incyte: Consultancy.

Cytotoxicity assays are essential for the testing and development of novel immunotherapies for the treatment of cancer. We recently described a novel cytotoxicity assay, termed the Matador assay, which was based on marine luciferases and their engineered derivatives. In this study, we describe the development of a new cytotoxicity assay termed ‘Matador-Glo assay’ which takes advantage of a thermostable variant of Click Beetle Luciferase (Luc146-1H2). Matador-Glo assay utilizes Luc146-1H2 and D-luciferin as the luciferase-substrate pair for luminescence detection. The assay involves ectopic over-expression of Luc146-1H2 in the cytosol of target cells of interest. Upon damage to the membrane integrity, the Luc146-1H2 is either released from the dead and dying cells or its activity is preferentially measured in dead and dying cells. We demonstrate that this assay is simple, fast, specific, sensitive, cost-efficient, and not labor-intensive. We further demonstrate that the Matador-Glo assay can be combined with the marine luciferase-based Matador assay to develop a dual luciferase assay for cell death detection. Finally, we demonstrate that the Luc146-1H2 expressing target cells can also be used for in vivo bioluminescence imaging applications.

Intro: Hematopoietic stem-cell transplant (HSCT) recipients are considered to be at high risk for poor outcomes following COVID-19 infection given their co-morbidities and immunosuppression. Sharma et al published the CIBMTR observational report data that showed that recipients of allogeneic HSCT who contract COVID-19 have poor overall survival with a 30-day mortality of 32%. That being said, there have been relatively few studies that look into the effect of COVID-19 on HSCT recipients in the setting of in vivo T cell depletion protocols. With the increased use of post-transplant cyclophosphamide (PTCy) based GVHD prophylaxis regimens for our match related and match unrelated HSCT recipients since 2018 at our institution, we are interested to see if our COVID-19 outcomes differ from those published in the CIBMTR report. Methods: This is a single institution retrospective analysis evaluating outcomes of HSCT recipients who were diagnosed with COVID-19 between March 2020 and April 2021. Patients 18 years or older who underwent HSCT and subsequently contracted COVID-19 were included in the data collection. Demographic data including age, type of hematologic malignancy, conditioning regimen, GVHD prophylaxis, date of COVID-19 infection, with pre- and post-COVID-19 infection labs were obtained. Our primary endpoint in this retrospective analysis was non-relapse mortality within 30 days of COVID-19 diagnosis. Results: There were 21 patients at our institution who had undergone HSCT and subsequently contracted COVID-19. The most common primary disease types were acute lymphoblastic leukemia (33.3%), acute myeloid leukemia (23.8%), and myelodysplastic syndrome (19.0%). The median age of our patient population was 53 years (range, 24-66). 6 of the patients received match related allografts. 7 received cells from match unrelated donors. 7 received cells from haploidentical donors. 1 patient had received an autologous stem cell transplant. Of the remaining 20 allo-HSCT recipients, 14 of them (70.0%) received myeloablative conditioning regimens, whereas 6 (30.0%) received reduced intensity or non-myeloablative regimens. Our GVHD prophylaxis regimens were PTCy/Tacro/MMF (12 pts, 60.0%) and Tacro/MTX (8 pts, 40.0%). Patient demographics and outcomes are found on Tables 1 and 2. Our patients were diagnosed with COVID-19 a median 469 days post-transplant, with 8 patients (38.1%) diagnosed with COVID-19 within 1 year of transplant. 11 of the patients (52.4%) received steroids following their diagnosis with COVID-19. Of the 20 allo-HSCT recipients with confirmed COVID-19 infection, 1 passed away 20 days after the diagnosis was made. This gives us a 5.0% case fatality rate attributable to COVID-19 in our population in our allo-HSCT population. 16 of the 20 patients were symptomatic at the time of diagnosis (80.0%). 7 of the 20 patients (35.0%) were hospitalized for a median of 7 days (range, 5-17 days), with 2 requiring ICU level of care. The one patient who passed away tested positive for COVID-19 177 days post-transplant and was hospitalized approximately 7 days after diagnosis, where he was intubated on hospital day 4 and ultimately passed away on hospital day 13. The patient had received Tacro/MTX for GVHD prophylaxis. Discussion: Although this is a small sample size, our data suggests that our allo-HSCT recipients who contracted COVID-19 have had generally good short-term outcomes. Our study is limited by the small number of patients who got infected with COVID-19, particularly those within 1-year post-transplant. Furthermore, we acknowledge it is difficult to claim that the PTCy based GVHD prophylaxis regimens for our HSCT recipients were solely responsible for their improved outcomes since 40% of allo-HSCT recipients did not get PTCy for GVHD prophylaxis. However, we believe it would be valuable to evaluate in a prospective analysis. We are currently evaluating if a COVID-19 diagnosis has any effect on long term transplant related complications and outcomes in this population. Figure 1 Figure 1. Disclosures Chaudhary: Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy ; Celldex: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pancella: Consultancy; Oncotartis: Consultancy; Athelas: Consultancy, Current holder of stock options in a privately-held company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company. Yaghmour: Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau.

Background: The use of post-transplant cyclophosphamide (PTCy)/tacrolimus/mycophenolate mofetil (MMF) for GVHD prophylaxis has improved outcomes in haploidentical hematopoietic cell transplantation (haplo-HCT). PTCy is now being evaluated in matched-related (MRD) and matched-unrelated (MUD) allo-HCT. Previous studies demonstrated improved GVHD-free/relapse-free survival (GRFS) when PTCy was combined with two immunosuppressive agents and PTCy has also been associated with better relapse-free survival (RFS) as demonstrated in De Jong et al 2019, though only one immunosuppressive agent was used. Currently, there is limited published data comparing outcomes using PTCy/tacrolimus/MMF to standard MRD/MUD GVHD prophylaxis of methotrexate (MTX)/tacrolimus. The importance of studying this comparison may help to improve GVHD outcomes in MRD and MUD allo-HCT. Methods: We retrospectively analyzed adult patients at USC Norris Cancer Hospital (age ≥ 19) who received allo-HCT from 2018 to 2020. The primary end-points assessed were incidence and severity of 1-year aGVHD and cGVHD. Secondary end-points included day+100 mortality, 1-year overall survival (OS), 1-year RFS, 1-year transplant-related mortality (TRM), and 1-year GRFS, defined as grade 3-4 acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death in the 1-year post-HCT period. Results: A total of 65 adult MRD and MUD allo-HCT recipients and 53 haplo-HCT patients were reviewed. Of the MRD/MUD patients evaluated, approximately 51% (n = 33) were female and 49% (n = 32) were male. The age range was 20-69 years old (median = 46), and the most common diseases included ALL (46%), AML (31%), MDS (11%), and others (i.e. lymphoma, aplastic anemia (AA), myelofibrosis) (12%). 34% (n = 22) of patients received PTCy on D+3 and D+4 with tacrolimus/MMF/ on D+5 as GVHD prophylaxis and 66% (n = 43) of patients received MTX/tacrolimus on D+1, +3, +6, and +11 as GVHD prophylaxis. All haplo-HCT patients received standard PTCy/tacrolimus/MMF. Stem cell source was primarily PBSC except in HLH and AA patients. The PTCy group had more MUD allo-HCT (64%), degree of antigen mismatch (56%), and median age of 50.5 years compared with the MTX group at 44%, 47%, and 44 years respectively. 70% in the MTX group received MAC compared with 45% in the PTCy group. The haplo group had similar demographics to the MTX group. The mean CD34 cell doses in the PTCy, MTX, and haplo groups were 4.87, 5.36, and 7.24x106 cells/kg respectively. Incidences of total GVHD, aGVHD, and aGVHD grade 3 or 4 in the PTCy group were 55%, 50%, and 4.5% respectively compared with 65%, 35%, and 7% in the MTX group, though not significant. The haplo group had 68%, 55%, and 1.9% respectively. Incidence of total cGVHD and cGVHD requiring systemic therapy in the PTCy group was 4.5% and 0% respectively compared with 30% (p = .02) and 23% (p =.01). The haplo group had 13% and 1.9% respectively. Day+100 mortality, 1-year OS, 1-year RFS, 1-year TRM, and 1-year GRFS in the PTCy group were 0%, 80%, 60%, 0%, and 64% respectively compared with 7%, 88%, 90%, 7.3%, and 59%. The haplo group had 3.8%, 86%, 89%, 14%, and 66%. In a univariate analysis, factors significantly associated with GVHD were disease status (p = .0.12) and CD34 dose (p = 0.015) and antigen mismatch (p = 0.04) was associated with increased mortality. Discussion: Our results demonstrate improved overall and extensive cGVHD outcomes in the PTCy group and thus an improvement in 1-year GRFS. Furthermore, incidence and severity of 1-year cGVHD in this group are improved when compared with previously reported outcomes. 1-year GRFS reported in De Jong et al 2019 was 45% and 1-year GRFS reported for all groups in our study is higher at 66%, 64%, and 59% for the haplo, PTCy, and MTX groups respectively. Although this was not significant, it may be clinically meaningful given the significant improvement in extensive GVHD and improvement in all other secondary end-points except 1-year OS and RFS. Furthermore, the PTCy group had a higher percentage of mismatched antigens yet demonstrated superior outcomes. 1-year OS and RFS were superior in the MTX group however this is likely due to sample size differences. The improved extensive cGVHD and GRFS outcomes observed using PTCy/tacrolimus/MMF in the MRD/MUD setting should continue to be evaluated and currently there is an ongoing prospective, randomized study to further investigate. Disclosures Yaghmour: Jazz: Consultancy, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Speakers Bureau; Alexion: Consultancy, Speakers Bureau; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION: Hispanic patients with acute lymphoblastic leukemia (ALL) are historically known to have poor outcomes compared to non-Hispanic patients. Our institution, LAC-USC (LA County Hospital/University of Southern California) has shown a complete remission rate of 96% with use of pegasparagase at 2000 IU/m2 using the USC ALL regimen (based on CCG-1882) for patients aged 17-55 years with a 7-year OS of 51% reported in 2014. The modified USC ALL regimen now uses a single dose of cytarabine rather than fractionated doses and uses a single dose 3g/m 2 methotrexate compared to 1g/m 2 (2.5g/m 2 if T-cell) D1, 15 in original USC ALL regimen to improve compliance, while consolidation was increased to six cycles allowing for PEG holidays to improve toxicity (Table 1). Since our institution takes care of a large population of Hispanic patients with ALL, we now report outcomes in Hispanic and Non-Hispanic patients using the modified USC ALL regimen in the era of novel agents like blinatumomab and inotuzumab. METHODS : This retrospective, single institution chart review included adults >18 years old with newly diagnosed Ph-negative ALL (2016-2020). Primary objectives were 3-year over-all survival (OS), event-free survival (EFS), disease-free survival (DFS). Secondary objectives were complete remission/complete remission with incomplete recovery (CR/CRi), minimal residual disease (MRD) by flow cytometry, descriptive statistics of patients who were stratified into Hispanic and non-Hispanic cohorts and evaluated using Fisher's exact test. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p-value ≤0.05 was significant. RESULTS: 121 Ph-negative patients were reviewed. 87 were Hispanic patients (HP) and 34 non Hispanic patients (NHP). Median ages were 39 and 35 years (p=0.51) and median BMI were 29 and 26.9 kg/m 2 (p=0.42), respectively. There were about equal males and females in HP while NHP had 70.6% males compared to 29.4% females (p=0.076). Both HP and NHP were mainly of the Ph-negative ALL subtype, 50.6% vs 47.1%, followed by Ph-like, 25.4% vs 20.6%, respectively (p=0.884). Majority of the population were unfavorable risk by NCCN karyotypic risk stratification, 55.9% in HP compared to 56.0% in NHP (p=0.99). Over-all, no significant difference between baseline characteristics in both cohorts. After induction 1: CR/CRi was 85.9% in HP and 73.4% in NHP (p=0.09). MRD negativity by flow cytometry in HP was 41.4% compared to 26.4% in NHP (p=0.13). After induction 2: 83% of HP were in CR/CRi compared to 80% in NHP (p=0.99) and MRD negativity by flow was 35.6% in HP compared to 32.4% in NHP (p=0.73). Blinatumomab was given in 33.3% of HP and 32.3% of NHP (p=0.92) while only 5.7% of HP and 2.9% of NHP received inotuzumab (p=0.99). 34.5% of HP underwent allogenic hematopoietic stem cell transplant (allo-HSCT) versus 26.5% in NHP (p=0.40). 3-year OS was 92.2% in HP versus 67.4% in NHP (p=0.004). 3-year DFS was 92.8% in HP versus 60.7% in NHP (p=0.003). 3-year EFS was 54.1% in HP versus 32.3% in NHP (p=0.02) (Table 2). Rate of relapse for HP and NHP were 23.4% vs 29.4% (p=0.74) while rate of mortality for HP and NHP were 7.5% vs 28% (p=0.015), respectively. No clear difference in grade 3/4 PEG toxicities were found in HP and NHP except more hypertriglyceridemia in HP (p=0.019). CONCLUSIONS: We present comparison of outcomes in Hispanic and non-Hispanic patients using our modified USC ALL pediatric-based regimen in an era of novel agents. Our data shows significantly better outcomes in Hispanic patients compared to non-Hispanic patients [OS (92.2% vs 67.4%, p=0.004), DFS (92.8% vs 60.7%, p=0.003) and EFS (54.1% vs 32.3%, p=0.02)]. This study demonstrates that given equal access to care (eg. receiving blinatumomab, allo-HSCT), outcomes in HP with Ph-negative ALL are not worse, but rather superior to those in NHP. Utilization of novel immunotherapy like blinatumomab in the salvage setting to achieve deeper molecular response and increased utilization of haploidentical allo-HSCT have likely contributed to reducing ethnic disparities in Hispanic ALL patients. Future studies are needed to validate these findings with larger patient populations. Figure 1 Figure 1. Disclosures Chaudhary: Oncotartis: Consultancy; Pancella: Consultancy; Moderna: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Athelas: Consultancy, Current holder of stock options in a privately-held company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Douer: Jazz: Consultancy; Amgen: Consultancy, Speakers Bureau; Adaptive: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Speakers Bureau; Servier: Consultancy, Speakers Bureau. Yaghmour: Takeda: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Alexion: Speakers Bureau; BMS: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Jazz: Speakers Bureau.

Intro: Acute and chronic graft versus host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplant (allo-HSCT). Post-transplant cyclophosphamide (PTCy), alongside mycophenolate mofetil (MMF) and tacrolimus (TAC), is known to be effective in reducing cGVHD in haploidentical (HI) transplant recipients. Results of the prospective randomized HOVON-96 trial in recipients of matched related (MRD) and unrelated donors (MUD) showed that PTCy leads to a reduction in risk of severe cGVHD. However, the role of PTCy in HSCT with acute lymphoblastic leukemia (ALL) remains controversial. The purpose of this study is to evaluate outcomes using PTCy, TAC, and MMF for GVHD prophylaxis in MRD and MUD stem cell recipients who have ALL. Methods: This study is a retrospective analysis of adult patients (aged 19 and up) with ALL who received MRD, MUD, and HI HSCT at USC Norris Cancer Hospital from 2018 to 2020. The primary end-points were to compare incidence of aGVHD and cGVHD within the first year post-transplant after receiving MRD, MUD, and HI donor transplants. Secondary end-points were day +100 mortality, 1-year overall survival (OS), relapse free survival (RFS), transplant-related mortality (TRM), and GRFS within the same subgroups. Kaplan-Meier curves and Log Rank tests were used to evaluate a difference in outcomes. Results: A total of 46 patients with ALL who received HSCTs were identified and separated based on type of GVHD prophylaxis received: 7 (15.1%), 16 (34.8%), and 23 (50.0%) in the PT-Cy, HI and standard groups respectively. Demographic data can be found on Table 1 attached. Baseline characteristics among all groups were similar with the exception of the more Human Leukocyte Antigen (HLA) mismatch (p Discussion: In this single institution analysis, we observed that patients with ALL who underwent HI transplant had the best outcomes. Moreover, patients receiving MUD/MRD HSCT with use of PTCy had a non-significant reduced incidence of aGVHD and cGVHD, though had significantly worse 1-year OS, RFS, and GRFS when compared to standard GVHD prophylaxis or patients receiving HI transplants. We believe this is likely a multifactorial process. However, the increased percentage of mismatched antigens in the MUD transplants (p=0.19) and higher proportion of patients receiving less than 5.0 x 10^6 CD34+ cells/kg (p=0.02) in the PTCy group is important to acknowledge and can also be contributing to their poorer outcomes. We pose the idea that the use of PTCy may also lead to a reduced graft vs. leukemia effect in this population and thus a higher incidence of relapse and worse outcomes overall. A larger, prospective study should be considered to take into account these factors to better determine if patients with ALL could benefit from PTCy for GVHD prophylaxis. Figure 1 Figure 1. Disclosures Chaudhary: Celldex: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company; Pancella: Consultancy; Oncotartis: Consultancy; Athelas: Consultancy, Current holder of stock options in a privately-held company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Yaghmour: Takeda: Consultancy, Speakers Bureau; Astellas: Speakers Bureau; Alexion: Speakers Bureau; BMS: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Agios: Consultancy, Speakers Bureau; Jazz: Speakers Bureau.

INTRODUCTION: The use of pediatric inspired regimens in adolescent young adults and adults has improved outcomes in acute lymphoblastic leukemia (ALL). CALGB 10403 was prospectively tested in patients 17-39 years, showing a 3-year event free survival of 59%. In 2007, our institution reported outcomes after incorporating peg-asparaginase (PEG) dosed at 2000mg/m 2 in induction for patients aged 17- 55 years. This regimen was well tolerated and resulted in a complete remission (CR) rate of 96%. In 2014, we reported experience with the use of multiple doses of PEG (CCG-1882) for newly diagnosed ALL adult patients, with a 7-year overall survival (OS) of 51%. Since our last report, the USC ALL regimen consisting of 2 induction phases, has been further modified with a goal of maintaining good outcomes, and improving compliance and toxicities. Fractionated doses of cytarabine were changed to a single dose, the methotrexate dose of 1g/m 2 (2.5 g/m 2 if T-cell) given D1,15 of intensification phases was changed to 3g/m 2 (B and T cell) given in single doses, and consolidation was increased to six cycles allowing for PEG holidays (Table 1). Moreover, the approval and incorporation of novel agents such as blinatumomab and inotuzumab also changed outcomes in ALL. Therefore, this study reports an update of outcomes since further modification of the USC ALL pediatric-based regimen in the era of novel agents. METHODS: This is a retrospective review which included adults aged >18 with newly diagnosed Philadelphia negative ALL between 2016 and 2020 treated at USC/Norris Cancer Center and Los Angeles County Hospital (LAC). Primary objectives were over-all survival (OS), event-free survival (EFS), disease-free survival (DFS) at 3 years, and secondary outcomes were complete remission/complete remission with incomplete recovery (CR/CRi) rates and minimal residual disease (MRD) by flow cytometry. OS, DFS, EFS were reported through Kaplan Meier curves and Log-rank tests. Two-sided p value ≤0.05 was significant. RESULTS: 121 patients with Ph-negative ALL were identified (49.6% Ph-negative B-ALL, 24% Ph-like B-ALL, 0.8% B cell lymphoblastic leukemia (LBL), 9.1% T cell ALL, 4.1% T cell LBL, 4.1% early T-cell, 5.8% mixed phenotype acute leukemia (MPAL). Median age at diagnosis was 38.5 years and maximum age of patient to receive pegasparagase during induction 1 was 63 years. 71.9% Hispanic, 15.7% white, 9.9% Asian, 2.5% African American. 57.9% males, 42.1% females. 3.4% were favorable, 4.2% intermediate, 54.6% unfavorable and 37.8% unknown by karyotypic risk stratification. Median BMI was 28.9 kg/m 2.54.6% had hepatic steatosis either on history or imaging and 5.1% had CNS disease pre-induction. Post-induction 1, 81.4% of patients achieved CR/CRi and 50% MRD negative. Post induction 2, 82.2% achieved CR/CRi, 67.7% MRD flow negative. Post-consolidation 1, 90.9% were MRD flow negative. 33.1% subsequently received blinatumomab for MRD positive disease, 5% given inotuzumab for relapsed disease, 32.2% underwent allogenic hematopoietic stem cell transplant (allo-HSCT). Median number of pegasparagase doses received during treatment was 2, rate of relapse and mortality was 27.3% and 13% respectively. Median OS and DFS were not reached but median EFS was 35 months. 3-year OS was 85.3%, when stratified according to MRD post induction 2; 3-year OS was 91.7% for MRD positive patients and 91.2% for MRD negative patients (p=0.55). 3-year DFS was 83.2%; 88.2% for MRD positive patients and 97.4% for MRD negative patients (p=0.22). 3-year EFS was 47.3%; 51.3% for MRD positive patients and 50.6% for MRD negative patients (p=0.49) (Tables 2-3). Use of pegasparagase resulted in grade 3/4 toxicities including hypersensitivity (4.1%), transaminitis (21.5%), pancreatitis (5.4%), hypertriglyceridemia (49.5%), hypofibrinogenemia (45.5%), hyperbilirubinemia (21.5%) and thrombosis (16.5%). CONCLUSIONS: Pediatric-based modified USC ALL induction regimen led to a high 3-year OS (85.3%), DFS (83.2%) and EFS (47.3%) with predictable toxicity and compares favorably with original USC ALL regimen, CALGB 10407, GRAALL 2005 and USC/MSKCC regimen. Interestingly, MRD positivity after induction 2 did not adversely affect OS, DFS or EFS, which is likely due to incorporation of blinatumomab and inotuzumab. These agents could have allowed for deeper remissions allowing Ph-negative patients with residual disease to receive allo-HSCT. Figure 1 Figure 1. Disclosures Chaudhary: Oncotartis: Consultancy; Pancella: Consultancy; Moderna: Current equity holder in publicly-traded company; Celldex: Current equity holder in publicly-traded company; TCR2: Current equity holder in publicly-traded company; Allogene: Current equity holder in publicly-traded company; Athelas: Consultancy, Current holder of stock options in a privately-held company; Angeles Therapeutics: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties: Cell therapy . Douer: Servier: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Adaptive: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Speakers Bureau; Jazz: Consultancy. Yaghmour: Novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Alexion: Speakers Bureau; Astellas: Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Jazz: Speakers Bureau; Agios: Consultancy, Speakers Bureau.

Success of immunotherapeutic approaches using genetically engineered antibodies and T cells modified with chimeric antigen receptors (CARs) depends, among other things, on the selection of antigen binding domains with desirable expression and binding characteristics. We developed a luciferase-based assay, termed Malibu-Glo Assay, which streamlines the process of optimization of an antigen binding domain with desirable properties and allows the sensitive detection of tumor antigens. The assay involves a recombinant immunoconjugate, termed Malibu-Glo reagent, comprising an immunoglobulin or a non-immunoglobulin based antigen binding domain genetically linked to a marine luciferase. Malibu-Glo reagent can be conveniently produced in mammalian cells as a secreted protein that retains the functional activity of both the antigen binding domain and the luciferase. Moreover, crude supernatant containing the secreted Malibu-Glo reagent can directly be used for detection of cell surface antigens obviating the laborious steps of protein purification and labeling. We further demonstrate the utility of Malibu-Glo assay for the selection of optimal single chain fragment variables (scFvs) with desired affinity characteristics for incorporation into CARs. In summary, Malibu-Glo assay is a fast, simple, sensitive, specific and economical assay for antigen detection with multiple applications in the fields of antibody engineering, antibody humanization and CAR-T cell therapy.

Chimeric Antigen Receptor-T (CAR-T) cell immunotherapy has produced dramatic responses in hematologic malignancies. One of the challenges in the field is the lack of a simple assay for the detection of CARs on the surface of immune effector cells. In this study, we describe a novel luciferase-based assay, termed Topanga Assay, for the detection of CAR expression. The assay utilizes a recombinant fusion protein, called Topanga reagent, generated by joining the extra-cellular domain of a CAR-target in frame with one of the marine luciferases or their engineered derivatives. The assay involves incubation of CAR expressing cells with the Topanga reagent, a few washes and measurement of luminescence. The assay can detect CARs comprising either immunoglobulin- or non-immunoglobulin-based antigen binding domains. We further demonstrate that addition of epitope tags to the Topanga reagent not only allows its convenient one step purification but also extends its use for detection of CAR cells using flow cytometry. However, crude supernatant containing the secreted Topanga reagent can be directly used in both luminescence and flow-cytometry based assays without prior protein purification. Our results demonstrate that the Topanga assay is a highly sensitive, specific, convenient, economical and versatile assay for the detection of CARs.

Background Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,β-didehydrophenylalanine (∆Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated. Methods The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy. Results and conclusions The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250 μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans. General significance The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.

Purpose: Kaposi sarcoma–associated herpes virus (KSHV)–associated primary effusion lymphomas (PEL) have extremely poor prognosis when treated with conventional chemotherapy. KSHV-encoded viral FLICE-inhibitory protein (vFLIP) K13 binds to the IkappaB kinase (IKK) complex to constitutively activate the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. The molecular chaperone HSP90 is a component of the IKK complex and is required for its activity. Experimental Design: We have analyzed the effect of HSP90 inhibitors on the survival and proliferation of PEL cells and on the activity of the NF-κB pathway. Results: We show that BIIB021, a purine scaffold–based orally administrable HSP90 inhibitor, shows preferential cytotoxicity toward PEL cells as compared with non-PEL cells. The cytotoxic effect of BIIB021 against PEL was associated with induction of cell-cycle arrest and apoptosis. BIIB021 blocked the expression of a number of cellular proteins involved in the regulation of cell cycle and apoptosis. BIIB021 also blocked constitutive NF-κB activity present in PEL cells, in part, by blocking the interaction of vFLIP K13 with the IKK complex subunits. In a xenograft model of PEL, BIIB021 significantly reduced tumor growth. Conclusion: BIIB021 blocks constitutive NF-κB activity in PEL and shows preferential antitumor activity against PEL in vitro and in vivo. BIIB021 may be a promising agent for treatment of PEL. Clin Cancer Res; 19(18); 5016–26. ©2013 AACR.

Increased risk of fungal diseases in immunocompromised patients, emerging fungal pathogens, limited repertoire of antifungal drugs and resistance development against the drugs demands for development of new and effective antifungal agents. With greater knowledge of fungal metabolism efforts are being made to inhibit specific enzymes involved in different biochemical pathways for the development of antifungal drugs. Chitin synthase is one such promising target as it is absent in plants and mammals. Nikkomycin Z, a chitin synthase inhibitor is under clinical development. Chitin synthesis in fungi, chitin synthase as a target for antifungal agent development, different chitin synthase inhibitors isolated from natural sources, randomly synthesized and modified from nikkomycin and polyoxin are discussed in this review.

In the present work, we investigated the antifungal activity of two de novo designed, antimicrobial peptides VS2 and VS3, incorporating unnatural amino acid α,β-dehydrophenylalanine (ΔPhe). We observed that the low-hemolytic peptides could irreversibly inhibit the growth of various Candida species and multidrug resistance strains at MIC(80) values ranging from 15.62 μM to 250 μM. Synergy experiments showed that MIC(80) of the peptides was drastically reduced in combination with an antifungal drug fluconazole. The dye PI uptake assay was used to demonstrate peptide induced cell membrane permeabilization. Intracellular localization of the FITC-labeled peptides in Candida albicans was studied by confocal microscopy and FACS. Killing kinetics, PI uptake assay, and the intracellular presence of FITC-peptides suggested that growth inhibition is not solely a consequence of increased membrane permeabilization. We showed that entry of the peptide in Candida cells resulted in accumulation of reactive oxygen species (ROS) leading to cell necrosis. Morphological alteration in Candida cells caused by the peptides was visualized by electron microscopy. We propose that de novo designed VS2 and VS3 peptides have multiple detrimental effects on target fungi, which ultimately result in cell wall disruption and killing. Therefore, these peptides represent a good template for further design and development as antifungal agents.

Kaposi sarcoma–associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with γ-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.

Kaposi sarcoma–associated herpesvirus (KSHV), also known as human herpesvirus 8, is the etiologic agent of Kaposi sarcoma (KS), an angioproliferative lesion characterized by dramatic angiogenesis and inflammatory infiltration. In this study, we report that expression of chemokine CCL20, a potent chemoattractant of dendritic cells and lymphocytes, is strongly induced in cultured cells either by KSHV infection or on ectopic expression of viral FLICE inhibitory protein K13. This induction is caused by transcriptional activation of CCL20 gene, which is mediated by binding of the p65, p50, and c-Rel subunits of the transcription factor nuclear factor–κB (NF-κB) to an atypical NF-κB–binding site present in the CCL20 gene promoter. The CCL20 gene induction is defective in K13 mutants that lack NF-κB activity, and can be blocked by specific genetic and pharmacologic inhibitors of the NF-κB pathway. CCR6, the specific receptor for CCL20, is also induced in cultured cells either by KSHV infection or on K13 expression. Finally, expression of CCL20 and CCR6 is increased in clinical samples of KS. These results suggest that KSHV and K13-mediated induction of CCL20 and CCR6 may contribute to the recruitment of dendritic cells and lymphocytes into the KS lesions, and to tumor growth and metastases.

Click reaction approach toward the synthesis of two sets of novel 1,2,3-triazolyl linked uridine derivatives 19a–19g and 21a–21g was achieved by Cu(I)-catalyzed 1,3-dipolar cycloaddition of 5′-azido-5′-deoxy-2′,3′-O-(1-methylethylidene)uridine (17) with propargylated ether of phenols 18a–18g and propargylated esters 20a–20g. Structure of one of the representative compound 19d was unambiguously confirmed by X-ray crystallography. Chitin synthase inhibition study of all these compounds 19a–19g and 21a–21g was carried out to develop antifungal strategy. Compounds 19d, 19e, 19f, and 21f were identified as potent chitin synthase inhibitors by comparing with nikkomycin. Compounds 19a, 19b, 19c, 19d, 21a, and 21b showed good antifungal activity against human and plant pathogens. Compounds 19a, 19b, 19f, 21c, 21f, and 21g were identified as lead chitin synthase inhibitors for further modifications by comparing results of inhibition of growth, % germ tube formation and chitin synthase activity.

EDAR plays a key role in the process of ectodermal differentiation via activation of the NF-kappaB pathway. We present evidence that EDAR also represses Lef-1/beta-catenin-dependent transcription and this ability is defective in EDAR mutants associated with anhidrotic ectodermal dysplasia. While IKK1/IKKalpha and IKK2/IKKbeta are required for EDAR-induced NF-kappaB activation, they are dispensable for its ability to repress Lef-1/beta-catenin-dependent transcription. In contrast, NIK is not involved in EDAR-induced NF-kappaB activation or Lef-1/beta-catenin transcriptional repression. As Lef-1/beta-catenin pathway controls the expression of EDAR ligand, ectodysplasin-A (EDA), our results point to a negative feedback regulation of EDA-EDAR axis.

The human herpes virus 8 (HHV8)-encoded viral FLICE inhibitory protein (vFLIP), also known as K13, is known to activate the NF-kappaB pathway, a property not shared by other vFLIPs. Previous studies have demonstrated that HHV8 vFLIP K13 interacts with several cellular signaling proteins involved in NF-kappaB activation, such as receptor-interacting protein, NF-kappaB-inducing kinase, IkappaB kinase (IKK) 1, IKK2, and NF-kappaB essential modulator (NEMO). In this report we have used cell lines deficient in the above proteins to investigate the mechanism of NF-kappaB activation via HHV8 vFLIP K13. We demonstrate that receptor-interacting protein and NF-kappaB-inducing kinase are dispensable for vFLIP K13-induced NF-kappaB DNA binding and transcriptional activation. On the other hand, vFLIP K13-induced NF-kappaB DNA binding activity is significantly reduced, although not absent, in cells deficient in IKK1, IKK2, and NEMO. Furthermore, vFLIP K13-induced NF-kappaB transcriptional activity is only weakly present in IKK1-deficient cells and almost completely absent in those deficient in IKK2 and NEMO. HHV8 vFLIP K13-induced NF-kappaB activation in IKK1- and IKK2-deficient fibroblasts could be rescued by wild type but not by the kinase-inactive mutants of IKK1 and IKK2, respectively. Consistent with the above results, vFLIP K13-induced NF-kappaB activation could be effectively blocked by chemical inhibitors of the kinase activity of IKK1 and IKK2. Thus, a cooperative interaction of all three subunits of the IKK complex is required for maximal NF-kappaB activation via HHV8 vFLIP K13. Selective inhibitors of the IKK1 kinase activity may have a role in the treatment of disorders caused by abnormal NF-kappaB activation by HHV8 vFLIP K13.

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases. The protective effect of HHV8 vFLIP is associated with its ability to activate the nuclear factor-kappa B (NF-kappaB) pathway and is missing in the vFLIP encoded by equine herpes virus 2 that lacks this activity. Inhibition of the NF-kappaB pathway by IkappaB superrepressor, lactacystin, MG132, arsenic trioxide, and phenylarsine oxide reverse the protection against growth factor withdrawal-induced apoptosis conferred by HHV8 vFLIP. HHV8 vFLIP up-regulates the expression of Bcl-x(L), an antiapoptotic member of the Bcl2 family, which is a known target of the NF-kappaB pathway. Collectively, the above results suggest that HHV8 vFLIP-induced NF-kappaB activation may contribute to cellular transformation seen in association with HHV8 infection by preventing the apoptosis of cells destined to die because of growth factor deprivation.

It is important to understand the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death. Defects in apoptosis are implicated in both tumorigenesis and drug resistance, and these defects are cause of chemotherapy failures. These studies should explain the relationship between cancer genetics and treatment sensitivity, and should enable a more rational approach to anticancer drug design and therapy. Lung cancer is a major cause of cancer deaths throughout the world. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) represent the two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. The role of apoptosis regulation in lung cancer with major focus on the differential sensitivities of the major subtypes is reviewed.

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-kappaB and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling. Whereas TRAF3 and -6, IKK1/IKKalpha, IKK2/IKKbeta, and NEMO/IKKgamma are involved in XEDAR-induced NF-kappaB activation, XEDAR-induced JNK activation seems to be mediated via a pathway dependent on TRAF3, TRAF6, and ASK1. Deletion and point mutagenesis studies delineate two distinct regions in the cytoplasmic domain of XEDAR, which are involved in binding to TRAF3 and -6, respectively, and play a major role in the activation of the NF-kappaB and JNK pathways. Taken together, our results establish a major role of TRAF3 and -6 in XEDAR signaling and in the process of ectodermal differentiation.

We have isolated a novel member of the TNFR family, designated TAJ, that is highly expressed during embryonic development. TAJ possesses a unique cytoplasmic domain with no sequence homology to the previously characterized members of the TNFR family. TAJ interacts with the TRAF family members and activates the JNK pathway when overexpressed in mammalian cells. Although it lacks a death domain, TAJ is capable of inducing apoptosis by a caspase-independent mechanism. Based on its unique expression profile and signaling properties, TAJ may play an essential role in embryonic development.

Caspase-8 is the most proximal caspase in the caspase cascade and possesses a prodomain consisting of two homologous death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins and is independent of their role as cell death proteases. A point mutant in the first DED of caspase-8 can block JNK activation induced by several death domain receptors. Inhibition of JNK activation blocks apoptosis mediated by caspase-10, Mach-related inducer of toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of this pathway in the mediation of caspase-induced apoptosis.

Remarkable structural and functional similarities exist between theDrosophila Toll/Cactus/Dorsal signaling pathway and the mammalian cytokine-mediated interleukin-1 receptor (IL-1R)/I-κB/NF-κB activation cascade. In addition to a role regulating dorsal-ventral polarity in the developing Drosophilaembryo, signaling through Drosophila Toll (dToll) activates the nonclonal, or innate, immune response in the adult fly. Recent evidence indicates that a human homologue of the dToll protein participates in the regulation of both innate and adaptive human immunity through the activation of NF-κB and the expression of the NF-κB–controlled genes IL-1, IL-6, and IL-8, thus affirming the evolutionary conservation of this host defense pathway. We report here the cloning of two novel human genes, TIL3 and TIL4 (Toll/IL-1R–like-3, -4) that exhibit homology to both the leucine-rich repeat extracellular domains and the IL-1R–like intracellular domains of human andDrosophila Toll. Northern analysis showed distinctly different tissue distribution patterns with TIL3 expressed predominantly in ovary, peripheral blood leukocytes, and prostate, and TIL4 expressed primarily in peripheral blood leukocytes and spleen. Chromosomal mapping by fluorescence in situ hybridization localized the TIL3 gene to chromosome 1q41-42 and TIL4 to chromosome 4q31.3-32. Functional studies showed that both TIL3 and TIL4 are able to activate NF-κB, though in a cell type–dependent fashion. Together with human Toll, TIL3 and TIL4 encode a family of genes with conserved structural and functional features involved in immune modulation.

Benzoyl phenyl urea, a class of insect growth regulator's acts by inhibiting chitin synthesis. Carvacrol, a naturally occurring monoterpenoid is an effective antifungal agent. We have structurally modified carvacrol (2-methyl-5-1-methylethyl] phenol) by introducing benzoylphenyl urea linkage. Two series of benzoylcarvacryl thiourea (BCTU, 4a-f) and benzoylcarvacryl urea (BCU, 5a-f) derivatives were prepared and characterized by elemental analysis, IR, H-1 and C-13 NMR and Mass spectroscopy. Derivatives 4b, 4d, 4e, 4f and 5d, 5f showed comparable insecticidal activity with the standard BPU lufenuron against Dysdercus koenigii. BCTU derivatives 4c, 4e and BCU 5c showed good antifungal activity against phytopathogenic fungi viz. Magnaporthe grisae, Fusarium oxysporum, Dreschlera oryzae; food spoilage yeasts viz. Debaromyces hansenii, Pichia membranifaciens; and human pathogens viz. Candida albicans and Cryptococcus neoformans. Compounds 5d, 5e and 5f showed potent activity against human pathogens. Moderate and selective activity was observed for other compounds. All the synthesized compounds were non-haemolytic. These compounds have potential application in agriculture and medicine. (C) 2012 Elsevier Ltd. All rights reserved.

The increase in the prevalence of multi drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis case demonstrates the urgent need of discovering new promising compounds with antimycobacterial activity. As part of our research program and with a aim of identifying new antitubercular drug candidates, a new class of 2-(trifluoromethyl)-6-arylimidazo[2,1- b ][1,3,4]thiadiazole derivatives has been synthesized by both conventional as well as microwave assisted method and evaluated for their in vitro antitubercular activity against M . tuberculosis H 37 Rv. Moreover, various drug-likeness properties of new compounds were predicted. Seven compounds from the series exhibited good activity with MIC in range 3.12–1.56 μg/ml. The present study suggests that compounds 6b , 6c , 6d , 6e and 6f may serve as promising lead scaffolds for further generation of new anti-TB agents.

Myeloma cells are dependent on IL6 for their survival and proliferation during the early stages of disease, and independence from IL6 is associated with disease progression. The role of the NF-κB pathway in the IL6-independent growth of myeloma cells has not been studied. Because human herpesvirus 8-encoded K13 selectively activates the NF-κB pathway, we have used it as a molecular tool to examine the ability of the NF-κB pathway to confer IL6 independence on murine plasmacytomas. We demonstrated that ectopic expression of K13, but not its NF-κB-defective mutant or a structural homolog, protected plasmacytomas against IL6 withdrawal-induced apoptosis and resulted in emergence of IL6-independent clones that could proliferate long-term in vitro in the absence of IL6 and form abdominal plasmacytomas with visceral involvement when injected intraperitoneally into syngeneic mice. These IL6-independent clones were dependent on NF-κB activity for their survival and proliferation but were resistant to dexamethasone and INCB018424, a selective Janus kinase 1/2 inhibitor. Ectopic expression of human T cell leukemia virus 1-encoded Tax protein, which resembles K13 in inducing constitutive NF-κB activation, similarly protected plasmacytoma cells against IL6 withdrawal-induced apoptosis. Although K13 is known to up-regulate IL6 gene expression, its protective effect was not due to induction of endogenous IL6 production but instead was associated with sustained expression of several antiapoptotic members of the Bcl2 family upon IL6 withdrawal. Collectively, these results demonstrate that NF-κB activation cannot only promote the emergence of IL6 independence during myeloma progression but can also confer resistance to dexamethasone and INCB018424.

Expression of A20, a negative regulator of the NF-κB pathway, is frequently lost in several subtypes of Hodgkin and non-Hodgkin lymphoma. We report that A20 is expressed in Kaposi sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma cell lines, and its expression correlates closely with the expression of KSHV-encoded viral FLICE inhibitory protein K13. Ectopic expression of K13 induced A20 expression through NF-κB-mediated activation of A20 promoter. In turn, A20 blocked K13-induced NF-κB activity and up-regulation of proinflammatory cytokines CCL20 and IL-8 in a negative feedback fashion. Both the N-terminal deubiquitinating domain and the C-terminal zinc finger domain of A20 were involved in the inhibition of K13-induced NF-κB activity. Overexpression of A20 blocked K13-induced IκBα phosphorylation, NF-κB nuclear translocation, and cellular transformation. Consistent with the above, K13-induced IκBα phosphorylation and NF-κB transcriptional activation were enhanced in A20-deficient cells. Finally, A20 was found to interact physically with K13. Taken collectively, these results demonstrate that K13 is a key determinant of A20 expression in KSHV-infected cells, and A20 is a key negative regulator of K13-induced NF-κB activity. A20 might serve to control the inflammatory response to KSHV infection and protect KSHV-infected cells from apoptosis.

Axl is an oncogenic receptor tyrosine kinase that plays multiple roles in tumorigenesis and metastasis of many cancers. This study is the first to demonstrate that Axl is induced in Kaposi sarcoma and Kaposi sarcoma herpesvirus (KSHV) transformed endothelial cells. Conditionally, expression of one KSHV latency protein vFLIP induces Axl expression in endothelial cells. This induction can be blocked by nuclear factor-κB inhibitor, consistent with the known vFLIP mechanism of action. KS cell lines lacking KSHV also have elevated Axl expression, which probably resulted from hypomethylation of AXL promoter. Axl activation activates downstream phosphoinositol-3 kinase signaling, and Axl knockdown by siRNA impairs phosphoinositol-3 kinase signaling. Furthermore, Axl knockdown inhibits KS cell growth and invasion. To explore the potential for translation of these findings, we generated monoclonal antibodies to block the biologic functions of Axl. MAb173, which induces receptor degradation, showed activity in vitro to inhibit KS cell invasion. Moreover, in vivo xenograft studies with KS cells with or without KSHV infection showed that MAb173 reduced tumor growth, increased tumor cell apoptosis, and markedly decreased Axl protein level in tumors. Axl thus has a potential role in KS pathogenesis and is a candidate for prognostic and therapeutic investigations.

P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.

Kaposi's sarcoma herpesvirus oncoprotein vFLIP K13 is a potent activator of NF-kappaB and plays a key role in viral pathogenesis. K13 contains a putative TRAF-interacting motif, which is reportedly required for its interaction with TRAF2. The K13-TRAF2 interaction is believed to be essential for the recruitment of K13 to the I-kappaB kinase (IKK) complex and for K13-induced NF-kappaB and JNK activation. In addition, TRAF3 has been reported to be required for K13-induced NF-kappaB and JNK activation. We have re-examined the role of the TRAFs in K13 signaling and report that mutations in the putative TRAF-interacting motif of K13 have no deleterious effect on its ability to interact with the IKK complex or activation of the NF-kappaB pathway. Furthermore, endogenously expressed TRAF2 and TRAF3 do not interact with K13 and play no role in K13-induced NF-kappaB activation or its interaction with the IKK complex. Finally, K13 does not activate the JNK pathway. Our results support a model in which K13 bypasses the upstream components of the tumor necrosis factor receptor signaling pathway and directly interacts with the IKK complex to selectively activate the NF-kappaB pathway without affecting the JNK pathway. Selective NF-kappaB activation by K13 might represent a novel strategy employed by the virus to promote latency.

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.

The Drosophila nuclear factor-kappaB (NF-kappaB)-like transcription factor Relish is activated by an endoproteolytic cleavage step mediated by the Drosophila caspase Dredd. We have examined the contribution of the caspase cascade to NF-kappaB activation via TRAIL, a mammalian tumor necrosis factor family ligand that is a potent activator of caspases. Our results demonstrate that TRAIL activates NF-kappaB in two phases as follows: an early caspase independent phase and a late caspase dependent phase. The late phase of the TRAIL-induced NF-kappaB is critically dependent on caspase 8 and can be blocked by pharmacological and genetic inhibitors of caspase 8 activation, such as benzyloxycarbonyl-VAD-fluoromethyl ketone, benzyloxycarbonyl-IETD-fluoromethyl ketone, and small interfering RNA targeting caspase 8 and FADD. Whereas caspase 3 is required for TRAIL-induced apoptosis, it is not involved in TRAIL-induced NF-kappaB activation. The late phase of TRAIL-induced NF-kappaB activation involves caspase mediated cleavage of IkappaBalpha between Asp(31) and Ser(32) residues to generate an N-terminal truncated fragment that is degraded by the proteasome via the N-end rule pathway. Our results demonstrate that caspases play an evolutionarily conserved role as regulated entry points to the N-end rule pathway and in NF-kappaB activation in mammalian cells.

Infection with human herpes virus 8 (HHV8) has been associated with Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. HHV8 encodes for a viral FLICE-inhibitory protein (vFLIP), designated K13, which resembles the prodomain of caspase-8 in structure and has been shown to protect cells against death receptor-induced apoptosis in vitro and in vivo. In this report, we present evidence that HHV8 vFLIP also possesses the unique ability of transforming Rat-1 and Balb/3T3 fibroblast cells, which is not shared by other vFLIPs. Rat-1 cells expressing HHV8 vFLIP form colonies in soft agar and form tumors in nude mice. The transforming ability of HHV8 vFLIP is associated with the activation of the NF-kappaB pathway and is blocked by molecular and chemical inhibitors of this pathway. Our results suggest that vFLIP K13 has activity beyond its role as an inhibitor of death receptor signaling and may play a causative role in the pathogenesis of HHV8-associated malignancies. Furthermore, inhibitors of the NF-kappaB pathway may have a role in the treatment of malignancies linked to HHV8 infection.

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.

Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. PEL cells universally express interferon regulatory factor 4 (IRF4). We found that shRNA-mediated knockdown of IRF4 is toxic to PEL. Further, we observed that FDA-approved immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show selective cytotoxicity towards PEL in a panel of cell lines comprising 11 hematological malignancies. The cytotoxic effect of IMiDs towards PEL was observed within their physiologically achievable concentrations and involved the downregulation of IRF4 and MYC. Genome-wide microarray examination (illumina) of PEL cells treated with lenalidomide followed by Gene Set Enrichment Analysis (http://www.broadinstitute.org/gsea) revealed activation of interferon (IFN) signaling. Further, recombinant IFNs α, β and γ are very effective and selective against PEL in a panel of cell lines tested. However, blocking the IFN pathway using specific blocking antibodies did not block the anti-proliferative potential of IMiDs towards PEL suggesting that activation of IFN pathway is not responsible for the cytotoxicity of IMiDs against PEL. Ikaros family proteins IKZF1 and IKZF3 are transcription factors which play crucial roles in immunity and cell-fate decisions. Recently, it has been shown that IMiDs selectively degrade these transcription factors (Kronke et al., and Lu et al., Science, 2014). In PEL, IKZF1 is strongly downregulated upon treatment with IMiDs. Additionally, shRNA-mediated knockdown of IKZF1 is toxic to PEL. Cereblon (CRBN) is a direct cellular binding target of IMiDs. IMiDs have no significant effect on the expression of CRBN in PEL and shRNA-mediated knockdown of CRBN (shCRBN) is not toxic to PEL. However, all the anti-PEL effects of IMiDs were blunted in cells expressing shCRBN. CRBN is a component and substrate receptor for Culling-RING ubiquitin ligase complex (CRL) (Ito et al., Science, 2010) and CRL activity depends on NEDDylation (Soucy et al., Nature, 2009). MLN4924, small molecule NEDD8-activating enzyme inhibitor phenocopied the effects of shCRBN on the anti-PEL activities of IMiDs suggesting that CRBN and its CRL activity is essential for the anti-proliferative activity of IMiDs in PEL. MYC is a downstream target of IMiDs in PEL but the role of MYC on the activity of IMiDs is not known. While investigating the role of MYC in IMiDs activity against PEL we found that shRNA-mediated knockdown of MYC enhanced the cytotoxicity of IMiDs suggesting a potential synergism between IMiDs and MYC inhibition. BRD4 inhibitors have been shown to block MYC expression (Delmore et al., Cell, 2011; Mertz et al., PNAS, 2011). We observed that lenalidomide is synergistic with all the BRD4 inhibitors (JQ-1, IBET151 and PFI-1) tested in PEL. Combined treatment of IMiDs with JQ-1 at low doses resulted in comprehensive downregulation of MYC and significant increase in the number of cells undergoing apoptosis as observed by AnnexinV staining and PARP cleavage. Knocking down BRD4 using specific shRNA also enhanced the cytotoxicity of IMiDs towards PEL suggesting that the synergism observed between IMiDs and BRD4 inhibition may not be limited only to the BRD4 inhibitors used in our study. Furthermore, combined administration of lenalidomide and JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model. These results provide the basis for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL. Disclosures Gopalakrishnan: University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties. Chaudhary:University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

Background: This clinical trial addressed feasibility and safety of microtransplantation by HLA-mismatched allogeneic cellular therapy (HMMACT) in poor risk acute myeloid leukemia patients. A secondary objective was to estimate complete response rate, infections, GVHD incidence, and induction mortality. Methods: Patients with high risk AML were enrolled: 4 were > 75 yo, 3 with MLL+ ((t(6;11), 11+ and t(10;11)), 3 complex/del7, one FLT3+. Patients received induction chemotherapy with mitoxantrone (10 mg/m2 IV for 3 days) and cytarabine (150 mg/m2 IV for 7 days) and received HLA-mismatched GCSF mobilized PBSCs on day 9 or 10. Family donors were concurrently HLA typed and best available donor underwent GCSF mobilization (5 mg/kg SQ BID x 4 days) and leukapheresis after medical evaluation and safety testing. HLA partial mismatch patients (4- 5/10 antigen) were chosen. Apheresis cells were counted and analyzed by flow cytometry for CD34+ cells; CD3+, CD4+CD25+ and CD8 + cells; and CD3-CD56+ NK cells. Target CD3 cell dose was 1 x 108/kg per cycle; cells cryopreserved as for standard stem cell donors. Family donor HLA-mismatched GCSF mobilized peripheral blood cells were infused fresh on day 9 or 10 (up to day 16), 36-50 hours after end of cytarabine. Bone marrow biopsy was evaluated on day 14 and 28 for AML remission status, and T/NK cell number. Patients achieving a complete response proceeded to consolidation with cytarabine 0.5 -1.0 gram/m2 x 6 doses with fresh or cryopreserved HMMACT cells from their donor for 2 courses a month apart. Patients: 10 patients age 31 – 80 yo consented: 8 received allo cells; 1 screen failure (no haplo family member identified); 1 patient too ill after initiating chemo to collect donor. Eight patients received at least one course of HMMACT; 1 withdrew early due to AML progression, a second for poor performance status. Three patients had de novo AML; 5 patients had secondary AML. Six Patients had2 or more cycles of HMMACT: four achieved CR: Pt 1 and 4 received 3 cycles, Pt 2 and 6 received 2 cycles of cell infusions and all achieved CR. Pt 9 received 2 inductions with cells, and sustained a partial clinical remission for 6 months. Two Patients had 1 cycle of HMMACT:Pt 3: received 1 cycle cells, but response not assessed; Pt 8: received 1 cycle cells, and achieved CR 3+mos but declined further therapy. Safety: Acute reactions to cell infusions were minimal. Delayed reactions attributed to cell infusions included fever grade 1-2, rash grade 1-2, diarrhea grade 1-2 resolving in 7-10 days. Patients with prior MDS or refractory leukemia requiring 2 inductions had a longer time to ANC and platelet recovery. Feasibility: Two patients had no donor; 2 donors only collected enough for 2 cell infusions. Response: 5/8 pts receiving cells had CR/CRi (62.5%) lasting 3 to 10+ months. Conclusions: Although there were the usual significant complications of treating leukemia and infections, the cellular therapy itself was well-tolerated. Patients often had fevers in first week after cell infusions, or experienced transient rash and diarrhea in the same time period, which were self resolving in all cases and may reflect elimination of allogeneic cells by the patient’s immune system. No patient developed GVHD. Feasibility of obtaining family donors was as expected for this diverse US population. Infectious complications were low by AML treatment standards. Complete remission rates are encouraging, but not as durable as hoped, likely reflecting the fact that all of our patients had high risk cytogenetics in contrast to the prior published studies by Chinese investigators. The majority were elderly who would not otherwise be offered intensive therapy. Family donors achieved expected cell targets and tolerated mobilization/collection procedures well. All but one patient receiving 2 to 3 cycles achieved complete remission. Two elderly patients who received one or two cell infusions during induction sustained prolonged survival of 6 months in spite of no further therapy, one achieving a CR and the other a sustained PR. Neutrophil and platelet recoveries tended to follow the pattern of the patient's prior secondary leukemia or myelodysplastic disorder. Once patients achieved complete remission however, recovery of blood counts was relatively rapid on further cycles of consolidation. We are now assessing Treg, T and NK cell phenotype of patient and donor cells by flow cyAtometry as biological correlates. Disclosures Chaudhary: University of Southern California: Inventor on a patent application relevant to this work filed to US patent office (No. 62/031,053). Patents & Royalties.

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-kappaB, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-kappaB or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.

Aging of the hematopoietic system in humans is associated with increased incidence of myeloid malignancies. Epigenetic machinery such as DNA methylation is known to change with age, and is disproportionately impacted by recurrent genetic mutations in acute myeloid leukemia (AML). We hypothesized that epigenetic changes in CD34+ hematopoietic stem and progenitor cells (HSPCs) may precede recurrent genetic changes in AML, and might be detected in normal aging HSPCs with increasing frequency. We also hypothesized that areas with increased variability in methylation may be hot-spots for the emergence of epigenetically distinct HSPC clones. In order to characterize these changes, we performed methylome-wide profiles of human HSPCs from different age groups (20-25 years (10), 40-45 years (10) and >60 years (9)).Adult HSPCs were obtained from Leukocyte Reduction System cones from healthy platelet donors. CD34+ cells were then isolated by Fluorescence Assisted Cell Sorting. 200 ng of DNA extracted from the CD34+ cells was processed using the Infinium Methylation 450k Beadchip (Illumina). Differentially methylated regions (DMRs) were identified using the bump hunting procedure of Jaffe (2011) to pool information across CpG loci into regions of consistent change and to quantify statistical significance. 893 differentially methylated regions (DMRs) varied linearly with age in HSPCs; a set of 31 such regions yielded an accurate predictor of age in lineage-sorted cells (N=48, Reinius et al., 2012) and whole blood (N=656, Hannum et al., 2011), with a root-mean-squared error of 5.3 years. While age-related lymphopenia has previously been reported, DNA methylation marks for lineage commitment (Houseman et al., 2012) were nearly uniform within our subjects’ CD34+ cells, and exhibited no relationship with age. However, regional summaries of methylation provided more accurate age predictions than specific CpG loci. We reasoned that differential variability at individual loci might be the cause. We thus investigated regions where methylation variability increased with age. Known imprinted clusters and allelically methylated regions (AMRs) identified by Fang (2012, PNAS) were disproportionately represented among these; 27% of known imprinting regions and 33.3% of allelically methylated regions in the genome coincided with at least one such region, while comprising only 0.3% of the genome and 0.7% of loci assayed. Among these, the H19 imprinting control region has been shown to crucially regulate long-term HSPC homeostasis in mice via IGF2, and the allelically methylated WT1/WT1-AS region on chromosome 11p is a highly recurrent hotspot for disordered methylation in AML, as well as sequential epigenetic defects in Wilms’ tumor. The allelically methylated vault RNA VTRNA2-1 (recently shown to predict survival in AML) on chromosome 5q, and the monoallelically expressed TP73 and DIRAS3 genes on chromosome 1p, were also sites of greater methylation variability with age in normal HSPCs. Wu et al. (1997) showed that loss of imprinting at H19/IGF2 is common in AML, seemingly conferring a selective metabolic advantage, and global loss of imprinting in mice leads to widespread tumorigenesis (Holm et al., 2005). Recurrent methylation aberrations in induced pluripotent stem cells favor imprinted clusters (Nazor, 2012), and epigenetic polymorphisms arise in these regions over time in cultured cells (Tanay et al, 2012). However, to our knowledge, ours is the first report of this type of heterogeneity with age in normal human adult HSPCs. Clonal hematopoiesis has previously been documented in healthy elderly adults (Levine 2012), and the majority of patients in the Cancer Genome Atlas (TCGA) AML study exhibited mutations in one or more genes regulating epigenetic machinery. We propose that increased epigenetic heterogeneity in aging HSPCs, particularly at regions with allele-specific methylation (such as H19/IGF2), may precede malignant evolution in some leukemias, and warrants further investigation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4886 Background: The R-CHOP regimen administered every three weeks has become the standard of care for treatment of patients with diffuse large B-cell lymphomas. Previous studies have not shown an advantage of dose dense (cycles given every 2 weeks) R-CHOP therapy. Granulocyte-macrophage colony stimulating factor (GM-CSF), in addition to stimulating hematopoetic recovery augments dendritic cell numbers and promotes antigen presentation, induces immune recognition of tumor cells, causes antibody dependent cytotoxicity, and upregulates activity of rituximab. In a Phase II study, we analyze the efficacy and tolerability of R-CHOP given every 15 days with GM-CSF administered on days 3 through 13 of each cycle. Methods: All patients were treated with Rituximab, 375 mg/m2; Cyclophosphamide, 750 mg/m2; Doxorubicin, 50 mg/m2; Vincristine, 1.4 mg/m2, all given IV on day 1; oral prednisone 100 mg given orally on days 1–5 and subcutaneous GM-CSF, 250 mg/m2 on days 3–13. Chemotherapy cycles were repeated every 15 days. Results: In this mid-study evaluation, thus far 36 patients with a median age of 53 (range 22–81) have been accrued. All patients were previously untreated and had CD20+ diffuse large B cell lymphoma. 26 (72%) patients had stage III (8) or IV (18) disease. Thirteen (36%) patients had bone marrow involvement. Twenty eight (77%) had elevated serum LDH levels, and 16 (44%) patients had an intermediate or high risk IPI score. Toxicities have been primarily hematologic with transient grade 3 or 4 neutropenia in 27 (74%) patients; grade 3 anemia in 6 (16%) patients; and grade 3 or 4 thrombocytopenia in 4 (11%) patients. In addition, 4 patients experienced neutropenic fever. Non-hematologic side effects of note were grade 3 mucositis/stomatitis in 2 patients; grade 3(n=1) and grade 4 (n=1) cardiac dysfunction in 2 patients. IgG, IgA, IgM levels were all decreased after 2 cycles of chemotherapy. Further cycles did not affect immunoglobulin levels. Of the 34 patients evaluable for response, 24 (71%) achieved complete remission, 7 (21%) achieved partial remission, 1 (3%) had stable disease, and 2 (6%) had progression of disease. Conclusion: The mid study evaluation confirms that R-CHOP given every 15 days with GM-CSF (sargramostim) administered on days 3 through 13 of each cycle provides promising response rates with tolerable toxicity. Disclosures: Tulpule: Genzyme, I am the PI on this study funded by Genzyme.: Research Funding.

Hem atopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport medlated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.

Several 1-(1-aryl-2-mercaptoacetylimidazole)-3-alkylcarbamides were synthesized and characterized by their sharp melting points, elemental analyses, and IR spectra. These substituted imidazolocarbamides possessed anticonvulsant activity, which was reflected by the 20-80% protection observed with these compounds against pentylenetetrazol-induced convulsions in mice. These substituted imidazolocarbamides selectively inhibited the in vitro oxidation of nicotinamide adenine dinucleotide (NAD)-dependent oxidations of pyruvate, alpha-ketoglutarate, beta-hydroxybutyrate, and NADH by rat brain homogenates. However, NAD-independent oxidation of succinate was not affected. The anticonvulsant activity possessed by 1-(1-aryl-2-mercaptoacetylimidazole)-3-alkylcarbamides had no relationship to their ability to inhibit cellular respiratory activity.

Several 1‐aryl‐3‐(2‐pyrimidyl)thiocarbamides and their corresponding cyclized 2‐arylimino‐3‐(2‐pyrimidyl)thiazolid‐4‐ones were synthesized and characterized by their sharp melting points and elemental analyses. These thiocarbamides and thiazolidones possessed anticonvulsant activity against pentylenetetrazol‐induced convulsions and potentiated pentobarbital‐induced hypnosis in mice. Most of these thiocarbamides and thiazolidones selectively inhibited nicotinamide adenine dinucleotide (NAD)‐dependent oxidation of pyruvate, where the use of added NAD decreased the degree of inhibition. The NAD‐independent oxidation of succinate, on the other hand, remained unaltered. The anticonvulsant activity of thiocarbamides and thiazolidones was unrelated to their ability to inhibit the respiratory activity of rat brain homogenates during oxidation of sodium pyruvate. Cyclization of thiocarbamides to the corresponding thiazolidones in general enhanced their CNS depressant and enzyme inhibitory effectiveness.

Several 1-(1-aryl-2-mercaptoacetylimidazole)-3-alkylcarbamides were synthesized and characterized by their sharp melting points, elemental analyses, and IR spectra. These substituted imidazolocarbamides possessed anticonvulsant activity, which was reflected by the 20-80% protection observed with these compounds against pentylenetetrazol-induced convulsions in mice. These substituted imidazolocarbamides selectively inhibited the in vitro oxidation of nicotinamide adenine dinucleotide (NAD)-dependent oxidations of pyruvate, alpha-ketoglutarate, beta-hydroxybutyrate, and NADH by rat brain homogenates. However, NAD-independent oxidation of succinate was not affected. The anticonvulsant activity possessed by 1-(1-aryl-2-mercaptoacetylimidazole)-3-alkylcarbamides had no relationship to their ability to inhibit cellular respiratory activity.

Eight 5-(3,4-methylenedioxyphenyl)-3-arylaminomethyl-I,3,4-oxadiazole-2-thiones were synthesized, characterized by their sharp melting points, elemental analyses, and IR spectra, and evaluated for anticonvulsant activity. All substituted oxadiazole-2-thiones possessed anticonvulsant activity, which was reflected by their ability to provide 10–70% protection against pentylenetetrazol-induced convulsions in mice at 100 mg/kg ip. These compounds inhibited in vitro nicotinamide adenine dinucleotide (NAD)-dependent oxidation of pyruvate, α-ketoglu-tarate, and NADH by rat brain homogenates as well as NAD-independent oxidation of succinate by rat brain homogenates. Antiproteolytic activity of these substituted oxadiazole-2-thiones was reflected by their ability to inhibit trypsin hydrolysis of bovine serum albumin. These results indicated that the inhibition of cellular respiration and antiproteolytic activity of these substituted oxadiazole-2-thiones is not the biochemical basis for their anticonvulsant activity.