Intensive Multiagent Chemotherapy Research Articles

Pre-clinical activity of targeting the PI3K/Akt/mTOR pathway in Burkitt lymphoma.

Though outcomes for pediatric Burkitt lymphoma (BL) have improved significantly in recent decades with intensive multi-agent chemotherapy and the addition of rituximab, chemotherapy resistance remains a significant impediment to cure following relapse. Activation of the PI3K/AKT pathway has been implicated in Burkitt lymphomagenesis and increased PI3K/AKT activation has been associated with worse outcomes in adults with aggressive B-cell non-Hodgkin lymphoma (B-NHL). Inhibitors of the PI3K/AKT pathway have been approved for the treatment of refractory indolent B-NHL and continue to be investigated for treatment of aggressive B-NHLs. We investigated the activation of the PI3K/AKT pathway in a cell line model of resistant BL and the ability to target this pathway with small molecule inhibitors in BL cell lines. We found that cell lines resistant to rituximab and chemotherapy exhibited increased activation of PI3K/AKT and that inhibition of AKT or PI3K results in in vitro anti-lymphoma activity. To investigate the role of PI3K/AKT activation on the efficacy of cytotoxic chemotherapy, we exposed cells to inhibitors in combination with chemotherapy and noted a synergistic increase in response to chemotherapy. Overall these findings highlight the role of PI3K/AKT in chemotherapy resistance in BL cells and may represent a tractable therapeutic target.

Abstract A23: Pterostilbene (3',5'-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro

Abstract Objective: Osteosarcoma (OS) is the most common primary malignant neoplasm of bone and mainly affects adolescents and young adults. Intensive multiagent chemotherapy and definitive surgery have dramatically improved its prognosis as well as functional outcome of affected limbs since the 1980s. However, survival rate of patients has remained around 70% for more than 30 years, and the prognosis remains especially poor in patients with metastatic disease or unresectable lesions. Thus, additional or alternative treatment strategy has long been desired for patients with such poor prognosis. Pterostilbene (PTE), a methyl ether of resveratrol derived from plants like grapes and blueberries, has recently attracted attention as a growing number of reports describe promising pharmacologic properties of antioxidative and antiaging effects and have shown its inhibitory effects against cancers including colon, oral cancer, and malignant lymphoma. However, there are very scarce reports about the effects of PTE against OS. Here, we show a very characteristic efficacy of PTE on human OS cells and propose its potential as a novel therapeutic intervention against OS. Materials and Methods: Effects of PTE on cellular growth were evaluated in human OS cell lines SaOS2, U2OS, and MG63 treated at concentrations ranging from 2 μM to10 μM (2, 5, 10 μM) for 48 hrs compared to nontreated cells by MTS assay. Additionally, we evaluated the effects of cotreatment of PTE with low-dose (0.2 μg/ml) of doxorubicin (DXR) against SaOS2 and U2OS. To investigate the efficacy of PTE on stemness, ability of spheroid formation in anchorage-independent 3D culture condition was evaluated in SaOS2 and U2OS cells treated by 10 μM PTE for 48 hrs and mRNA expression of stemness markers of Oct3 and CD44 was evaluated by RT-PCR at the same condition. Results: Treatment of PTE showed dose-dependent growth inhibitory effects on all cell lines and was especially significant on MG63 cells at the highest inhibition rate of 65% at the concentration of 10 μM for 48 hrs treatment. In contrast, PTE did not show growth inhibition on normal mesenchymal stromal cells, one of the candidates for OS cell of origin. Cotreatment of PTE with DXR indicated that PTE augmented the growth suppressive effects of DXR lifting the growth inhibition rate up to 515% from DXR alone against SaOS2 and U2OS (62-67% and 25-40%, respectively). PTE treatments decreased sphere-forming ability in both SaOS2 and U2OS cells in terms of sphere numbers and sphere sizes, and also decreased mRNA expression of stemness markers of Oct3 and CD44 in both cell lines. Discussion: Current results suggest that PTE not only inhibits cellular growth of OS but also suppresses stem cell phenotype which was defined by sphere-forming ability and mRNA expression of stemness markers. Mechanisms of stem cell phenotype suppression by PTE are still unknown, but it might be caused by either decrease or differentiation of stem cell subpopulation. Suppression of stem cell phenotype could lead to enhancing the efficacy of cytotoxic chemotherapeutic agents such as DXR. Previous reports suggested that antitumor activity of PTE against OS cells targeted the JAK2/STAT3 signaling pathways. The efficacy of PTE is cell-type dependent, possibly due to the mechanisms of cell death or cell metabolism. The molecular pathways involved in growth inhibition and stem cell phenotype suppression caused by PTE are under investigation using pathway inhibitors and metabolism modulators. Conclusion: PTE inhibits cellular growth and augments the efficacy of DXR against OS cells possibly through the suppression of stem cell characteristics represented by sphere-forming ability and mRNA expression of stemness markers. The current study suggests that PTE could be a possible candidate for a novel therapeutic intervention against OS. Citation Format: Shingo Kishi, Kanya Honoki, Shinji Tsukamoto, Hiromasa Fujii, Yumiko Kondo, Akira Kido, Toshifumi Tsujiuchi, Hiroki Kuniyasu, Yasuhito Tanaka. Pterostilbene (3',5'-dimethoxyresveratrol) inhibits cell growth and augments the efficacy of doxorubicin through stem cell phenotype suppression in osteosarcoma cells in vitro [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A23.

An Off-the-Shelf™ Fratricide-Resistant CAR-T for the Treatment of T Cell Hematologic Malignancies

T cell malignancies represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults for which there are currently no effective or targeted therapies. Despite intensive multi-agent chemotherapy regimens, fewer than 50% of adults and 75% of children with T-ALL survive beyond five years. For those who relapse after initial therapy, salvage chemotherapy regimens induce remissions in 20-40% of cases. Allogeneic stem cell transplant, with its associated risks and toxicities, is the only curative therapy.T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown great potential for inducing both remissions and even long-term relapse-free survival in patients with B cell leukemia and lymphoma7-9. Thus, a targeted therapy against T cell malignancies represents a significant unmet medical need. However, several challenges have limited the clinical development of CAR-T cells against T cell malignancies. First, the shared expression of target antigens between T effector cells and T cell malignancies results in fratricide, or self-killing, of CAR-T cells. Second, harvesting adequate numbers of autologous T cells, without contamination by malignant cells is, at best, technically challenging and prohibitively expensive. Third, the use of genetically modified CAR-T cells from allogeneic donors may result in life-threatening graft-vs.-host disease (GvHD) when infused into immune-compromised HLA-matched or mismatched recipients.We hypothesized that deletion of CD7 and the T cell receptor alpha chain (TRAC) using CRISPR/Cas9 in CAR-T targeting CD7 (UCART7) would result in the efficient targeting and killing of malignant T cells without significant effector T cell fratricide or induction of GvHD.To generate the CD7 CAR, the anti-CD7 single chain variable fragment (scFv) was created using commercial gene synthesis and cloned into the backbone of a 3rd generation CAR with CD28 and 4-1BB internal signaling domains. The construct was modified to express CD34 via a P2A peptide to enable detection of CAR following viral transduction. Human primary T cells were activated using anti-CD3/CD28 beads for 48 hours prior to bead removal and electroporation with CD7 gRNA, TRAC gRNA, and Cas9 mRNA. On day three, T cells were transduced with lentivirus particles encoding either CD7 CAR or CAR CD19 control and allowed to expand for a further 6 days. Transduction efficiency and ablation of CD7 and TRAC were confirmed by flow cytometry. Multiplex CRISPR/Cas9 gene-editing resulted in the simultaneous bi-allelic deletion of both CD7 and TRAC in 72.8%±1.92 of cells, as determined by both non-homologous end joining (NHEJ) and FACS analyses. To prevent alloreactivity, CD3+ CAR-T were removed from the product by magnetic depletion. Of particular importance is that by using two distinct methods for assessing “off-target” nuclease activity across the entire human T cell genome (Guide-seq and probe capture), we could only detect one gene, an intronic modification of RMB33, that was inappropriately targeted using this approach. No obvious genomic rearrangements were detected by these analyses.UCART7 effectively expanded and killed T-ALL cell lines (CCRF-CEM, MOLT3, and HSB2) and human primary T-ALL blasts in vitro. Next, we tested the capacity of UCART7 to kill primary T-ALL in vivo without xenogeneic GvHD. Considerable expansion of alloreactive T cells, severe GvHD (mean clinical GvHD score = 5.66), and a robust graft vs. leukemia effect were observed in recipients of WT T cells. In contrast, GvHD was completely absent, T cells were undetectable, and considerable tumor burden was observed in mice receiving TRACΔ T cells. Mice receiving UCART7, however, had no GvHD and, unlike UCART19 controls, effectively cleared primary human T-ALL in NSG mice.Fratricide-resistant and allo-tolerant ‘off-the-shelf’ UCART7 signifies a novel strategy for treatment of relapsed and refractory T-ALL and non-Hodgkin's T cell lymphomas without a requirement for autologous T cells and represents the first clinically feasible adoptive T cell therapy for T cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Targeting FLT3 Signaling in Childhood Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the second most common leukemia of childhood and is associated with high rates of chemotherapy resistance and relapse. Clinical outcomes for children with AML treated with maximally intensive multi-agent chemotherapy lag far behind those of children with the more common acute lymphoblastic leukemia, demonstrating continued need for new therapeutic approaches to decrease relapse risk and improve long-term survival. Mutations in the FMS-like tyrosine kinase-3 receptor gene (FLT3) occur in approximately 25% of children and adults with AML and are associated with particularly poor prognoses. Identification and development of targeted FLT3 inhibitors represents a major precision medicine paradigm shift in the treatment of patients with AML. While further development of many first-generation FLT3 inhibitors was hampered by limited potency and significant toxicity due to effects upon other kinases, the more selective second- and third-generation FLT3 inhibitors have demonstrated excellent tolerability and remarkable efficacy in the relapsed/refractory and now de novo FLT3-mutated AML settings. While these newest and most promising inhibitors have largely been studied in the adult population, pediatric investigation of FLT3 inhibitors with chemotherapy is relatively recently ongoing or planned. Successful development of FLT3 inhibitor-based therapies will be essential to improve outcomes in children with this high-risk subtype of AML.

Incidence and risk factors of bacterial and fungal infection during induction chemotherapy for high-risk neuroblastoma

ABSTRACTHigh-risk neuroblastoma is an aggressive childhood cancer with poor outcomes. Treatment begins with an induction phase comprised of intense multi-agent chemotherapy with the goal of maximally reducing tumor bulk. Given the high intensity of induction chemotherapy, neutropenic fever and infectious complications are common; however, the actual incidence is difficult to determine from clinical trial reports. We performed a retrospective review of infection-related complications in 76 children treated for high-risk neuroblastoma at Texas Children's Hospital. Medical records were reviewed for demographics, febrile neutropenia (FN) episodes, presence, and type of bacterial and fungal infections, and potential risk factors for infection. Fifty-seven percent of patients developed one or more serious bacterial or fungal infections during induction chemotherapy. Additionally, over 75% of patients had at least one admission for FN. Risk factors for developing any infection included female sex, MYCN amplification, and having Medicaid. Patients with external central venous catheters and those requiring parenteral nutrition had higher rates of bacteremia or fungemia. Each cycle, 50% were readmitted for either FN or infection. The overall burden of infectious complications was high, with 70% having two or more unplanned admissions for infection or FN. The incidence of febrile neutropenia and serious bacterial and fungal infections during induction chemotherapy for high-risk neuroblastoma is high. Most patients had at least two additional hospitalizations for infectious complications. Risk factors including female sex, MYCN amplification, payer status, and type of central access were associated with higher rates of infection in this cohort.Abbreviations: CLABSI Central line associated blood stream infection; CTCAE Common Terminology Criteria for Adverse Events; FN Febrile neutropenia; ANC Absolute neutrophil count; TPN Total parenteral nutrition.

Treatment of aggressive B-cell lymphomas.

Treatment of aggressive B-cell lymphomas.

QoL of pediatric-inspired compared to hyper-CVAD regimens for newly diagnosed AYA patients with Ph-ALL: A modeling analysis.

e22002 Background: The treatment of acute lymphoblastic leukemia (ALL) requires 2+ years of intensive multi-agent chemotherapy, with significant impact on quality of life (QoL). While treatment protocols exist for adolescent / young adult (AYA) patients, there is limited evidence on the QoL impact for patients treated with pediatric-inspired protocols, which include asparaginase, versus regimens such as Hyper-CVAD. Objective: To compare the QoL impact of pediatric-inspired vs Hyper-CVAD regimens in the 1st-line treatment of AYA patients with Ph- ALL using disease simulation. Methods: A 6-state Markov model was developed to simulate total life-years and quality-adjusted life-years (QALYs) for AYA ALL patients receiving 1st line pediatric-inspired or Hyper-CVAD regimens. Modeled health states were: no first complete remission (no treatment/non-response to 1st line), first complete remission, first relapse, second complete remission, second relapse, and death. Model inputs for CR, EFS, OS, AEs, and health utility were obtained from literature. Weibull function was fitted to extrapolate survival and QALY over time. Results: Compared with Hyper-CVAD, the pediatric-inspired protocol resulted in increased life-years and QALYs at years 5, 10 and through a lifetime horizon. Benefits were mainly driven by more favorable progression-free survival and OS of patients in pediatric-inspired protocols compared to hyper-CVAD. Conclusions: This disease simulation model suggests that pediatric-inspired protocols for newly diagnosed AYA patients with Ph- ALL may increase QoL, as measured by QALYs, compared to Hyper-CVAD. Future long-term follow-up studies are needed to confirm these findings. [Table: see text]

Polatuzumab Vedotin; Antibody Drug Conjugates (ADC, anti-CD79b-vc-MMAE, PV) Significantly Enhances in-Vitro Cell Death Against CD79b+ Burkitt Lymphoma (BL) /Primary Mediastinal Large B Cell Lymphoma (PMBL) and Improved Overall Survival Against Rituximab Sensitive/Resistant-BL NSG Xenograft Mice

Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over 30 years through the use of short and intense multi-agent chemotherapy, however, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 years, EFS,Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Miles/Cairo et al. BJH. 2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease.The anti-CD79b-vc-MMAE antibody drug conjugates (ADC, Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009; Dornan et al. Blood. 2009). More recently PV has been safe and well tolerated in adults with CD79b refractory NHL (Palanca-Wessels et al. Lancet Oncol.2015), However, PV preclinical activity against mature BL/PMBL is unknown.Objective: To determine the efficacy of the ADC (anti-CD79b-vc-MMAE) against CD79b+ PMBL in-vitro and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro/ in-vivo.Methods: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106P and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79B mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji and Raji4RH cells at 5x106 tumor cells/mouse as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system.Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death ( apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20µg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p=0.001 and p=0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p=0.004 and 0.006), Karpas1106P, 46.4±0.3 %vs 29.0±1.5% vs. 15.9±0.6% (p=0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p=0.01 and p=0.0005), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b antibody or IgG isotype control in Raji and Raji4RH ( 35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003 and 50 vs. 18 vs. 18.5 days, p=0.0001, 0.0001, respectively, Figure. A and B)Conclusion: Our preliminary data indicates thathu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in RTX sensitive/ and resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Furthermore, PV significantly increased survival in BL (RTX sensitive/ resistant) NSG xenografts compared to an equal dose of anti-CD79b alone. [Display omitted] DisclosuresCairo:Celgene: Research Funding.

Abstract 3838: Upregulation of DLEU1 significantly prolongs survival in a rituximab-treated DLEU1 knockout human Burkitt lymphoma (BL) xenograft NSG mouse model: DLEU1 may act as a tumor suppressor gene in pediatric BL

Abstract BACKGROUND: Burkitt lymphoma (BL) is the most common histological subtype of non-Hodgkin lymphoma (NHL) in children and adolescents (Cairo et al., Blood 2007; Miles/Cairo., BJHaem 2012). We identified that pediatric BL patients with chromosome 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al., Leuk 2009; Nelson/Cairo/Sanger et al., BJH 2009). Deleted in lymphocytic leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 region (Dave et al., NEJM 2006) and interacts with C-MYC, TUBB2C and UBR1 (Stelzl et al., Cell 2005). We have previously reported a regulated DLEU1 expression significantly associated with changes in BL proliferation in vitro (Lee/Cairo et al., AACR 2015). We hypothesize that DLEU1 may have function as a tumor suppressor gene; however, functions and mechanisms underlying DLEU1 expression in the BL are poorly understood. OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene in BL and therefore examined whether up-regulation of DLEU1 results in changes in survival following targeted immunotherapy. METHODS: DLEU1 stably overexpressing Raji BL cells (Lee/Cairo et al., ASH 2013) were transfected with a firefly luciferase expression plasmid, kindly provided by Laurence Cooper MD, PhD. Four- to six- week-old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice from The Jackson Lab. Tumor burden and tumor progression were monitored by bioluminescence imaging system. Mice were treated with either rituximab (30 mg/kg) or cyclophosphamide (25 mg/kg) by intraperitoneal injection at 7 day intervals. Survival rates were analyzed by the Kaplan-Meier method using the Prism Version 6.0 software. RESULTS: We observed that DLEU1 stably overexpressing Raji BL cells xenografted (GFP-DLEU1) mice had significantly extended survival compared to GFP control mice following treatment rituximab (59 vs 51 days, p<0.05, n = 8), cyclophosphamide (50.5 vs 37.5 days, p<0.0001, n = 12) and in combination treatment (60.5 vs 53 days, p<0.05, n = 12) whereas there were no significant differences in survival between WT and DLEU1-KO mice with PBS treatment. We observed that there were no significant differences of luciferase intensity in between GFP and GFP-DLEU1 mice following rituximab, cyclophosphamide or in combination treatment. CONCLUSIONS: We demonstrated that the upregulation of DLEU1 expression significantly improved the survival in DLEU1 overexpressing BL cells xenografted NSG mice following rituximab, cyclophosphamide and in combination treatment. Therefore, the up-regulation of DLEU1 expression in BL may in part result in sensitizing to chemoimmunotherapy induced survival and may result in a consideration of potential therapeutic strategies Citation Format: Sanghoon Lee, Changhong Yin, Tishi Shah, Janet Ayello, Erin Morris, Carmella van de Ven, Mitchell S. Cairo. Upregulation of DLEU1 significantly prolongs survival in a rituximab-treated DLEU1 knockout human Burkitt lymphoma (BL) xenograft NSG mouse model: DLEU1 may act as a tumor suppressor gene in pediatric BL. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3838.

Dexrazoxane Does Not Negatively Impact Survival Outcomes in Children and Young Adults with Acute Myeloid Leukemia (AML)

Introduction: Acute myeloid leukemia (AML) comprises 5% of pediatric cancers and reports 5 year event free survival (EFS) of approximately 50% and overall survival (OS) of 60-70% following intensive multi-agent chemotherapy. Anthracyclines have been the cornerstone in upfront treatment of AML since the 1960's and continue to be used today in upfront trials for AML. Anthracycline induced cardiotoxicity remains a significant contributor to late morbidity/mortality in children and young adults with AML. The cardioprotectant dexrazoxane (Zinecard) can be used as prophylaxis to diminish the risk for cardiomyopathy but whether it affects the risk of relapse in pediatric AML is unproven. Our institution adopted the use of dexrazoxane prior to administration of any non-liposomal anthracycline for all patients in 2011. We are therefore reporting the differences in cardiac and treatment outcomes in children and young adults with AML treated with and without dexrazoxane from 2008 to 2013.Methods: We performed a retrospective chart review of children ages 0 to 21 years who received their therapy for AML at the Children's Hospital of Wisconsin (CHW) between January 1, 2008 and December 31, 2013. This study was approved by the CHW institutional review board prior to our data collection. Descriptive statistics were used to describe the study population. Echocardiogram statistics were generated with each model based on the least squares means and then transformed back to its original unit. A log rank test was used to detect differences between populations. Based on number of patients, a hazard ratio of 3 will achieve 85% power to detect a significant difference between groups.Results: Forty-four patients with AML were treated at CHW during 2008 - 2013 with 28 (64%) receiving dexrazoxane cardioprotectant and 16 (36%) did not. The median age at diagnosis was 8.1 years (5 months - 21.7 years) and 55% (n=24) were female. Six (14%) patients underwent transplantation in first complete remission and 6 (14%) underwent transplantation following relapse. No patients had history of cardiac disease, cardiac surgery or hypertension prior to treatment.We identified no statistical difference in relapse free survival (RFS) p>0.40), EFS (p>0.48) or OS (p>0.53) between groups. However, there was a significant decrease in the ejection fraction (EF %) (p=0.0018) and a significant decrease in shortening fraction (SF %) (p=0.0108) trends over time in the non-dexrazoxane group compared to patients who received dexrazoxane (Figures 1 & 2).Conclusion: Utilization of the cardioprotectant dexrazoxane prior to anthracycline chemotherapy in pediatric patients with AML demonstrated no significant difference in RFS or OS relative to our institutional controls and appeared to improve cardiac function. Further studies are needed to confirm these findings. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

NOTCH1 Induces Differential Epigenomic Patterning and Genomic Organization in Fetal Liver- and Adult Bone Marrow-Derived Hematopoietic Progentiors

T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T-cell progenitors, characterized by activating NOTCH1 mutations in over 50% of children and adult cases. Although intensive multiagent chemotherapy achieves cure in most pediatric patients, the majority of adults succumb quickly to their disease. The basis for this divergence is likely multifactorial, but we sought in this study to investigate whether cell intrinsic features might contribute to the disparate biologies in pediatric and adult patients.In our prior abstract, we modeled pediatric and adult leukemias by transduction of hematopoietic stem/progenitor cells (HSPC) derived from mouse fetal liver (FL) and adult bone marrow (ABM) with activated NOTCH1 virus followed by transplantation into histocompatible recipient animals. We observed that whereas FL- and ABM-derived HSPC generate similar primary acute T-cell leukemias in terms of penetrance, latency, disease burden/distribution, and immunophenotype, FL leukemias exhibit much greater cycling activity than ABM leukemias, yet are dramatically impaired in their ability to propagate disease in secondary and tertiary recipients compared to ABM leukemias. Using a combination of gene expression profiling and in vitro culture assays, we attributed this differential behavior to NOTCH1-induced autocrine IGF signaling that is operative in FL, but not ABM-derived HSPC.Here we report that NOTCH1 mediates its effects on IGF1 in FL-derived HSPC directly by physical occupancy over the IGF1 promoter in a dimerization-dependent fashion. As well, increased NOTCH1 occupancy at the IGF1 promoter region in FL tissues is associated with reduced histone H3K27 trimethylation (a mark of transcriptionally silent chromatin), yet there is equivalent histone H3K4 trimethylation (a mark identifying transcriptionally active promoters) in both FL and ABM tissues, suggesting that NOTCH1 may be responsible for interconverting the IGF1 locus between active and inactive, but poised chromatin states. NOTCH1 occupancy is also associated with enhanced physical interactions between the IGF1 promoter region and distant genomic loci as revealed by circularized chromosome conformation capture (4C) assay and confirmed by chromosome conformation capture (3C) assay, including sites with H3K4 monomethylation (a mark of transcriptional enhancers) suggesting that NOTCH1 promotes "looping in" of distant enhancer elements that drive IGF1 expression in FL tissues. We conclude from these studies that NOTCH1 enacts differential, developmental stage-specific transcriptional programs by a combination of local epigenetic patterning and long-range genomic interactions. These findings support the notion that pediatric and adult T-ALL may potentially be regarded as related, but biologically distinct diseases, and that novel, age-specific therapies that exploit these differences may improve clinical outcomes. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2077: DLEU1 significantly alters programmed cell death in chemoimmunotherapy-treated TALENs-induced DLEU1 knockout and DLEU1 overexpressing Burkitt Lymphoma (BL): DLEU1 may act as a tumor suppressor gene in pediatric BL

Abstract BACKGROUND: Burkitt Lymphoma (BL) is the most common histological subtype of childhood and adolescent Non Hodgkin Lymphoma (Cairo et al, Blood 2007; Miles/Cairo, BJH 2012). Pediatric patients with BL with a chromosome 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leukemia 2009; Nelson/Cairo/Sanger et al, BJH 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene in the 13q14.3 (Dave et al, NEJM 2006) and it has been implicated in regulating programmed cell death in patients. Sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) have been developed for precision targeted genome editing in in vitro and in vivo (Sander et al, Nat Bio 2011). OBJECTIVES: We hypothesize that DLEU1 may act as a tumor suppressor gene and therefore investigated whether DLEU1 expression results in changes in gene expression profiles, cell viability, apoptosis induced by chemoimmunotherapy regimens in DLEU1 knockout and/or DLEU1 overexpressing Raji BL cells. METHODS: TALEN-mediated DLEU1 knockout (DLEU1-KO) and DLEU1 overexpressing Raji BL cells (DLEU1-OE) were treated with Rituximab (RTX) and/or Cyclophosphamide (CTX). MTS, Caspase assay, qRT-PCR, western blot, and gene expression profiling using Affymetrix HG-133+ 2.0 arrays were performed. RESULTS: A significant decrease of apoptosis in DLEU1-KO (32.5±11.8%, p<.05) and a significant increase in DLEU1-OE (9.7±5.5%, p<.05) were observed vs control. A total 2,501 differentially expressed genes were identified by gene expression profiling (>2 fold, p<.05) and 79.8% of genes including signaling pathways and anti-apoptosis related genes including ATK1, ikBa and Bcl-2 were upregulated in DLEU1-KO.. There was a significant decrease in apoptosis with RTX, CTX and in combination (0.85±0.05, 0.76±0.09, and 0.65±0.17, p<.05, respectively) in DLEU1-KO whereas a significant increase with RTX (11.5%, p<.05), CTX (13.9%, p<.05) in DLEU1-OE. A significant increase in cell proliferation was observed with RTX, CTX and combination (1.14±0.06, 1.1±0.06, and 1.09±0.02, p<.05) in DLEU1-KO whereas a significant decrease with RTX, CTX and in combination (0.92±0.01, 0.89±0.04, and 0.87±0.04, p<.05, respectively) in DLEU1-OE vs control. CONCLUSIONS: We demonstrate that 1) DLEU1 knockout resulted in significant changes of ikBa and Akt1 expression, inhibition of apoptosis and increase of viability in RTX and CTX treatment; 2) DLEU1 overexpression significantly inhibited cell viability and increased apoptosis in RTX and CTX treatment. We hypothesize that deletion of DLEU1 in pediatric BL may in part result in chemotherapy resistance secondarily to a loss of a tumor suppressor gene. Citation Format: Sanghoon Lee, Changhong Yin, Janet Ayello, Carmella van de Ven, Mitchell S. Cairo. DLEU1 significantly alters programmed cell death in chemoimmunotherapy-treated TALENs-induced DLEU1 knockout and DLEU1 overexpressing Burkitt Lymphoma (BL): DLEU1 may act as a tumor suppressor gene in pediatric BL. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2015-2077

Early results from Children’s Oncology Group (COG) ARST08P1: Pilot studies of cixutumumab or temozolomide with intensive multiagent chemotherapy for patients with metastatic rhabdomyosarcoma (RMS).

10015 Background: Data from a previous COG study (ARST0431) showed an early FFS improvement but no long-term survival benefit with intensive interval-compressed chemotherapy in metastatic RMS. ARST...

Pediatric mature B-cell non Hodgkin lymphoma treatment with LMB-96 protocol. The Children Cancer Hospital Egypt experience

Purpose: Burkitt lymphoma (BL) is a highly aggressive mature B-cell non-Hodgkin lymphoma (NHL) and is the fastest growing human tumor. The outcome of childhood NHL has improved steadily over the past decades through the use of intensive sequential multi-agent chemotherapy regimens. Methods: A retrospective study having all patients 18 years old or younger diagnosed with mature B cell NHL and treated at Children Cancer Hospital Egypt (CCHE). All children were treated according to the modified (LMB 96) protocol during the period between July 2007 and December 2012. Patients were followed up till June 2013. Results: Three hundred and seventy-seven patients were diagnosed with mature B cell NHL and received the LMB96 treatment protocol. The majorities were males (76.4%) with a median age of 5.3 years, and ranged from 0.1-18.0 years. The median follow-up period was 28.2 months (range 0.9-72 months). Burkitt lymphoma was the most predominant pathologic subtype (79.6%, n = 300), and abdominal mass as a primary site was the most common presentation (71.3%). Twenty seven patients (7.2%) were treated as group A, 268 (71.0%) as group B, and 82 (21.8%) patients as high risk group C. Seventy-one (18.8%) patients suffered adverse events. Major adverse events were early deaths in 17 patients (4.5%), death during induction chemotherapy seen in 18 patients (4.7%), and during maintenance therapy in 7 patients (1.8%), tumor progression in 19 patients (5.0%), and relapse in 10 patients (3.7%). Sixty-three patients (16.7%) died during the study period. The main causes of death were tumor lysis syndrome (TLS) in 25.3%, and severe sepsis during chemotherapy in 41.3% of the patients. The 3 years OS and EFS were 83.3% and 80.4% respectively for the whole groups of patients. OS and EFS were 100% for group A, and 87.5%±3.9% and 85.9±4.3% for group B. For group C BM + /CNS - patients, OS was 55.62%±15.8%, and EFS of 53.8%±15.6%. For BM + /CNS + patients, OS and EFS were 63.2%±21.76% and 57.9%±22.1% respectively. BM - /CNS + patients had OS 72.4%±18.8% and EFS 67.6%±19.7% at 36 months. Conclusion: TLS and chemotherapy related toxicity remains a major challenge affecting the outcome of pediatric mature B cell NHL. We identified bone marrow involvement as a risk factor affecting treatment outcome. Aggressive supportive care measures are mandatory to avoid unacceptable high toxicity related mortality.

Comparative Study of Obinutuzumab (GA101) Vs. Rituximab Against CD20+ rituximab-Sensitive and -Resistant Burkitt (BL) and Acute Lymphoblastic Leukemia (B-ALL): Potential Targeted Therapy in Patients with High Risk BL and Pre-B-ALL

Background: Aggressive non-Hodgkin lymphoma (NHL) represents >90% of all NHL that occur in children and adolescents. Among all NHLs, Burkitt Lymphoma (BL) is the most common NHL in children and adolescents and has an excellent prognosis (≥80% 5 yrs, EFS) following short but intense multi-agent chemotherapy (Cairo et al. Blood, 2007). Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-ALL) have a dismal prognosis, often associated with chemotherapy resistance and may require alternative therapeutic strategies (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013). Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe and well-tolerated and is associated with >90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Goldman/Cairo et al. Leukemia, 2013). Resistance to RTX, however, may predispose patients with CD20+ B-NHL/ALL to an increase risk of relapse and/or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012,). Obinutuzumab, a novel glycoengineered type II CD20 antibody, has been shown to enhance cell death and ADCC vs. RTX (Herter et al, Clinc Canc Res, 2013), and was recently approved by FDA and EMA for first line treatment of CLL in combination with chlorambucil.Objective: To evaluate anti-tumor activity of obinutuzumab vs RTX against RTX resistant and sensitive BL and pre-B-ALL tumor targets in-vitro and in-vivo in xenografted NSG mice.Methods: Raji (CD20+) and Loucy (T-ALL, CD20-), (ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany) and Raji-4RH (provided by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS. For in-vitro studies, tumor cells were incubated with 100 µg/ml obinutuzumab (supplied by Christian Klein, PhD, Roche Research & Early Development, Zurich), and/or RTX for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD and analysis by flow-cytometry. Loucy cells (CD20-) were used as the negative control. ADCC were performed with K562-IL-15-41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) at 20:1 effector: target ratio (E: T, n=3) using an europium release assay (Perkin-Elmer).The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy for in vivo evaluation by BLI. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice, bred in-house under pathogen free conditions, were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg, obinutuzumab (30 mg/kg), and RTX (30 mg/kg).Mice were xenografted with intravenous injections of Luc+ Raji, Raji4RH, U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were then injected every 7 days with the respective therapy for 8 weeks. Mice were monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system.Results: Obinutuzumab compared to RTX (100 mg/ml, 24hrs), significantly enhanced cell death in Raji 45.1±3.3% vs 32.7±6.8%, (p=0.005), Raji4RH 15.8±2.2% vs 2.1±1.5% (p=0.001) and U698-M 40.5±2.9 % vs 26.36±2.6% (p=0.001) n=6. Obinutuzumab vs RTX also elicited a significant increase ADCC with K562-IL15-41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% (p=0.001), Raji-4RH 40.0±1.6% vs 0.5±1.1%, (p=0.001), and U-698-M 70.0±6 % vs. 45.56± 0.1% (p=0.001) n=3.Further, we found that, in vivo, obinutuzumab was significantly more effective than RTX when administered at the same doses in BL (RTX resistant/sensitive) and pre-B-ALL xenografts. Overall survival in mice receiving 30 mg/kg of obinutuzumab was significantly increased when compared to mice receiving 30 mg/kg of RTX in BL; Raji (p=0.05), Raji4RH (p=0.024) and U698-M (p=0.03) (Figure1: A, B and C).Conclusion: Obinutuzumab significantly enhances cell death and NK mediates ADCC in sensitive and RTX resistant CD20+ B-NHL and B-ALL compared to RTX. These preliminary studies also demonstrate that RTX sensitive/resistant BL and pre-B-ALL xenografted mice display significantly increased survival when given 30 mg/kg of obinutuzumab and decreased tumor burden in BL and Pre-B-ALL xenografts compared to an equal dose of RTX. Obinutuzumab may be a novel agent to investigate as adjuvant therapy in patients with relapsed refractory CD20+ B-NHL and/or B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Intensified Chemotherapy for Older Patients with Acute Lymphoblastic Leukemia (ALL): A Phase II Study from the Dana Farber Cancer Institute (DFCI) ALL Consortium

▪Unlike the significant advances seen with intensive chemotherapy for pediatric acute lymphoblastic leukemia (ALL) over the last two decades, long-term outcomes among patients over age 50 remain poor, with median survival less than one year. This contrast has been attributed to high-risk chromosomal features, decreased compliance with and tolerance of effective therapies, and exposure to less intensive multi-agent regimens among adults with ALL. In recent years, more intensive chemotherapeutic paradigms, derived from pediatric protocols, have been studied in adult ALL. The purpose of the current study was to determine the efficacy of an intensified multi-agent approach, derived from a completed DFCI consortium pediatric regimen used in younger adults, in an older (age >50) population of patients with ALL. For this study, modifications of the pediatric regimen included incorporation of clofarabine in consolidation, adjustment to dose and scheduling of PEG asparaginase and steroids, as well as inclusion of stem cell transplant (SCT) for eligible patients. The primary endpoint was survival rate at 1 year, with the goal of improving from the 33% historical control to 53%.Adults, aged 51-75 years, with newly diagnosed ALL or lymphoblastic lymphoma, were eligible. During induction, patients received multi-agent chemotherapy with vincristine, prednisone, doxorubicin, and PEG asparaginase. Imatinib was instituted if cytogenetics confirmed the presence of the Philadelphia chromosome. Patients received prophylactic intrathecal therapy with induction, and those with CNS involvement underwent additional IT therapy. Prednisone was administered for 21 days for those aged less than 60 and for 7 days for those aged 60 and above. Following induction, cycle one of consolidation included treatment with clofarabine, prednisone, and PEG asparaginase. After induction and first consolidation course, eligible patients proceeded to allogeneic SCT. Patients without matched sibling donors could receive unrelated donor or cord blood transplants. While those eligible under age 60 could undergo ablative conditioning regimens, those 60 and older received reduced intensity regimens. Those not eligible for SCT, went on to receive CNS, consolidation and continuation phases of treatment, as per protocol, which incorporated treatment with cycles of vincristine, doxorubicin, 6-mercaptopurine, and dexamethasone. PEG asparaginase was incorporated into the induction, consolidation I, CNS phase, and consolidation II phases of therapy.As of the most current analysis, 30 patients have been enrolled. A total of 19 of 29 evaluable patients (66%) have achieved a complete remission (CR). Three patients were refractory to induction therapy, four discontinued treatment during induction due to toxicity, of which three died, and nine patients have experienced relapse following remission. Nine patients have undergone SCT. A total of 15 patients have died on study out of 27 evaluated, and the overall survival, calculated by the method of Kaplan and Meier, at one year was 62% [95% CI, 41%-77%] (Figure 1), while disease-free survival for the 18 patients who achieved a CR following induction therapy at one year was 77% [95% CI, 49%-90%]. In total, for evaluable patients with at least one year of follow-up, the proportion surviving at one year was 61% [two-sided 80% CI, 47-75%] (16/26), significantly higher than the historical rate (33% used for this analysis, one-sided 90% exact CI) among such patients. Overall survival is also shown for Ph+ and Ph- groups (Figure 2). The most common grade 3/4 toxicities included transaminitis and hyperbilirubinemia, cytopenias, hypophosphatemia, hyperglycemia, and neutropenic fever. The major toxicity of liver injury, thought related to PEG asparaginase, prompted an amendment to the protocol to reduce the dose. Additionally, PEG asparaginase administration was limited to only those with Philadelphia chromosome-negative disease, to decrease risk of severe hepatotoxcity in patients receiving concurrent imatinib and PEG asparaginase.These data suggest that intensive multi-agent chemotherapy is tolerable in older patients with ALL, and can result in improved outcomes when compared to historical data. Additional study of similarly intensive regimens, incorporating novel therapies and alternative formulations of asparaginase, are warranted in older populations of ALL. [Display omitted] [Display omitted] DisclosuresFathi:Seattle Genetics: Research Funding; Seattle Genetics: Advisory Board, Advisory Board Other. Stone:Novartis: Research Funding.

Molecular therapeutic approaches for pediatric acute myeloid leukemia.

Approximately two-thirds of children with acute myeloid leukemia (AML) are cured with intensive multi-agent chemotherapy. However, refractory and relapsed AML remains a significant source of childhood cancer mortality, highlighting the need for new therapies. Further therapy intensification with traditional cytotoxic chemotherapy in pediatric AML is not feasible given the risks of both short-term and long-term organ dysfunction. Substantial emphasis has been placed upon the development of molecularly targeted therapeutic approaches for adults and children with high-risk subtypes of AML with the goal of improving remission induction and minimizing relapse. Several promising agents are currently in clinical testing or late preclinical development for AML, including monoclonal antibodies against leukemia cell surface proteins, kinase inhibitors, proteasome inhibitors, epigenetic agents, and chimeric antigen receptor engineered T cell immunotherapies. Many of these therapies have been specifically tested in children with relapsed/refractory AML in Phase 1 and 2 trials with a smaller number of new agents under Phase 3 evaluation for children with de novo AML. Although successful identification and implementation of new drugs for children with AML remain a formidable challenge, enthusiasm for novel molecular therapeutic approaches is great given the potential for significant clinical benefit for children who do not have other curative options.

A multicenter phase II study incorporating high-dose rituximab and liposomal doxorubicin into the CODOX-M/IVAC regimen for untreated Burkitt's lymphoma

BackgroundDespite improvement with intensive multi-agent chemotherapy, 2-year progression-free survival (PFS) rates for adults with high-risk Burkitt's lymphoma (BL) remains <55%. Patients and methodsWe conducted a phase II trial for newly diagnosed classic BL utilizing liposomal doxorubicin (Adriamycin) in lieu of doxorubicin and incorporating intravenous rituximab (at 500 mg/m2 twice/cycle) into the CODOX-M/IVAC regimen. Correlative analyses included paired serum and cerebrospinal fluid (CSF) rituximab levels and close examination of cardiac function. ResultsAmong 25 BL patients, the median age was 44 years (23–70) and 4 patients were HIV positive. There were 20 high-risk and 5 low-risk patients. At baseline, 40% of high-risk patients had bone marrow involvement, 35% had bulky disease and 15% had central nervous system involvement. The overall response rate was 100% (complete remission 92%). At 34-month median follow-up, the 2-year PFS and overall survival (OS) rates for all patients were 80% and 84%, respectively (low-risk: both 100%; high-risk: 76% and 81%, respectively). Furthermore, the 2-year PFS, OS, and disease-specific survival (DSS) rates for high-risk, HIV-negative patients were 84%, 89% and 100%, respectively. Adverse events (AEs) appeared to be consistent with prior CODOX-M/IVAC data, although there were several grade 3 cardiac events noted (all declined ejection fraction without clinical symptoms). The mean serum rituximab levels at 24 h after cycles 1 and 3 for patients without relapse were 258 and 306 μg/ml, respectively, versus 131 and 193 μg/ml, respectively, for patients with early progression (P = 0.002 and 0.002, respectively). The mean CSF rituximab levels for all patients were 0.11 and 0.24 μg/ml, respectively, at cycle 1 (24/72 h), which equated to serum:CSF ratios of 0.05% and 0.20%, respectively. ConclusionsThe integration of rituximab into CODOX-M/IVAC for adult BL was feasible and tolerable, while changes in cardiac function warrant continued examination. This regimen was associated with excellent survival rates for HIV-negative BL. Further investigation of the predictive value of serum rituximab is needed. Clinicaltrials.gov NCT00392990.

Sequence-Specific Talens-Induced DLEU1 Gene Disruption Significantly Suppresses Apoptosis and Increases Phosphorylation Of AKT and Ikba In Pediatric Burkitt Lymphoma (PBL): DLEU1 May Act As a Tumor Suppressor Gene In PBL

BackgroundPediatric Burkitt Lymphoma (BL) is the most common histological subtype of Non Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJHaem, 2012). We previously identified, in a subset analysis, that children with BL and a 13q deletion, particularly 13q14.3, had significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leukemia, 2009; Nelson/Cairo/Sanger et al, BJHaem, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene on 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 has been implicated in regulating programmed cell death in patients by inducing TUBB2C and UBR1 expression. Sequence-specific Transcription Activator-Like Effector Nucleases (TALENs) technologies have been developed for precision targeted genome editing in in vitro and in vivo experiments with high efficiency as a new experimental and therapeutic tool (Sander et al, Nat Bio, 2011). We have previously reported TALENs mediated transient downregulation of DLEU1 in Raji cells (Figure 1), and demonstrated a decrease in the spontaneous apoptotic rate and an increase in cell proliferation in transiently DLEU1 down-regulated Raji cells (Lee/Cairo et al, ASH, 2012). ObjectivesWe hypothesize that DLEU1 may act as a tumor suppressor gene and therefore investigated whether the silencing of DLEU1 results in changes in proliferation, programmed cell death and associated signaling pathways induced by Rituximab and Cyclophosphamide alone and in combination in TALEN-mediated DLEU1 knockout Raji BL cells. MethodsA stable, single DLEU1 knockout clone (DLEU1 KO) was isolated from Raji cells transfected with DLEU1 TALENs (Lee/Cairo et al, ASH, 2012). Genomic DNA and total RNA were extracted using a DNA extraction kit (Promega) and Trizol (Invitrogen) for verification of endogenous DLEU1 gene disruption by TALENs. qRT-PCR was performed by CFX96 (Bio-rad) and the protein level was measured by western blotting. DLEU1 KO cells were plated (1x104 cells) into 48 or 96 well plates and then treated with Rituximab (0, 1, 10 and 100ug/ml) and/or Cyclophosphamide (0, 1, 5 and 10mM) and cell proliferation and apoptosis were measured by MTS (Promega) and Caspase 3/7 (Promega) assay. Statistical significance was determined by a one tailed paired Student t-test. ResultsDLEU1 KO (mono-allelic deletion) in Raji (BL) exhibited significantly reduced DLEU1 mRNA expression compared to mock control cells (83% reduction, p<0.006). There was significantly reduced expression of c-myc, TUBB2C and UBR1 mRNA (85%, p<0.001; 60%, p<0.04; 25%, p<0.006), respectively in DLEU1 KO compared to mock control cells. DLEU1-KO showed significant reduction of Caspase 3/7 activities (25% at 24hrs, p<0.007; 33% at 48hrs, p<0.03) and significant increase in cell proliferation (20% at 24hrs, p<0.04; 25% at 48hrs, p<0.03). DLEU1-KO showed a significant increase in expression of anti-apoptotic genes, Bcl-2, Bcl-xL and Mcl-1 mRNA (14%, p<0.03; 30%, p<0.04; 17%, p<0.04), respectively and a significantly a decrease in mRNA expression of pro-apoptotic genes, Bax and Bad (18%, p<0.04; 14%, p<0.04), respectively. There was a significant increase in phosphorylation of IkBa (2.6 fold, p<0.007) and Akt (1.5 fold, p<0.05) in DLEU1 KO. There was a significant reduction in Caspase-dependent apoptosis with RTX (15% reduction, p<0.05), CTX alone (24% reduction, p<0.03), and in combination (35% reduction, p<0.03) in DLEU1-KO compared to mock control. A significant increase in cell proliferation was observed with RTX (10% increase, p<0.04), CTX alone (9.4% increase, p<0.05), and combination (7.7% increase, p<0.02) in DLEU1-KO. RTX treated DLEU1-KO showed a significant increase in expression in Bcl-2 and Bcl-xL mRNA (1.3 fold, p<0.05 and 2.0 fold, p<0.02), respectively and a significant decrease in mRNA expression of Bax (1.2 fold, p<0.03). ConclusionsWe demonstrate that 1) TALENs mediated DLEU1 gene knockout resulted in downregulation of DLEU1 mRNA and consequently, inhibition of BL apoptosis and increase in cell proliferation, through several mechanism(s), and 2) the silencing of DLEU1 resulted in decrease in apoptosis in RTX, CTX alone and in combination treatment. We hypothesize that deletion of DLEU1 in childhood BL may result in chemotherapy resistance secondarily to a loss of a tumor suppressor gene. Disclosures:No relevant conflicts of interest to declare.▪

Exogenous Overexpression Of DLEU1 Significantly Induces Programmed Cell Death and Inhibits Cell Proliferation In Burkitt Lymphoma (BL) and Sensitizes Rituximab (RTX) Resistant BL Cells To RTX Induced Apoptosis

BackgroundPediatric Burkitt Lymphoma (PBL) represents over 40% of Non-Hodgkin Lymphoma (NHL) in children and adolescents (Cairo et al, Blood, 2007; Miles/Cairo, BJH, 2012) and the prognosis of PBL has dramatically improved over the past 30 years through the introduction of short intensive multi-agent chemotherapy. (Cairo et al, JCO, 2012). However, the success of this therapeutic approach has been compromised by significant chemotherapy-induced toxicity, requiring the need to identify a less toxic targeted therapy. We previously identified, in a multivariate analysis, that children with Burkitt Lymphoma (BL) and a 13q deletion had a significantly poorer outcome and inferior overall survival (OS) despite aggressive short, intensive multi-agent chemotherapy (Poirel/Cairo et al, Leuk., 2009; Nelson/Cairo/Sanger et al, BJH, 2009). Deleted in Lymphocytic Leukemia 1 (DLEU1) is a BL classifier gene and a target of c-Myc on the chromosome 13q14.3 region (Dave et al, NEJM, 2006). DLEU1 encodes for proteins of 72 residues and 19 interacting proteins including c-Myc, TUBB2C, RASSF1A and UBR1. We have previously suggested that DLEU1 may function as a tumor suppressor gene in transient DLEU1 overexpressed BL cells (Lee/Cairo, ASH, 2012). However, functions and mechanism(s) following dysregulation of DLEU1 expression in BL are poorly understood. ObjectivesWe proposed that DLEU1 may act as a tumor suppressor gene in PBL, and therefore investigated whether 1) overexpression of DLEU1 in BL cells results in changes in programmed cell death, cell proliferation and cell migration, and 2) sensitizes Rituximab resistant BL cells to apoptosis in PBL. MethodsA stable Raji cell line with DLEU1 overexpression (DLEU1-OE) was established from previous reported (Lee/Cairo et al, ASH, 2012) under G418 selection and all cells including Rituximab (RTX) resistant Raji BL cells (2R) (Barth et al, BJH, 2012) were cultured in RPMI with 10% FBS. Total RNA was isolated by Trizol (Invitrogen) and cDNA was synthesized by cDNA Synthesis Kit (Quantas). Quantitative RT-PCR was performed by CFX96 Real-time (Bio-rad), and MTS and Caspase 3/7 assays (Promega) were employed for measurement of cell proliferation and apoptosis. Statistical significance was performed by one-tailed Student t-test. For classic scratch-wound healing assay, wounded cells were created by scratching cells at 72 hours post-transient transfection with DLEU1 and images of wound areas were taken at 0 and 48 hours after scratching. For transient experiments using 2R, DLEU1 transient overexpressing Raji and 2R cells were treated with Rituximab (20ug/ml) for 48hours and cell proliferation and apoptosis assay were measured as above. ResultsThe expression of DLEU1 mRNA showed a significant increase in DLEU1 stable overexpressing Raji (DLEU1-OE) cells compared to mock control cells (9.0 fold increase, p<0.02). In comparing mRNA expression levels of DLEU1 network genes, there was significant decrease of RASSF1 and TUBB2C mRNA expression (1.2 fold reduction, p<0.02 and 1.3 fold reduction, p<0.03, respectively) in DLEU1-OE cells to mock control cells. DLEU1-OE cells showed a significant decrease in cell proliferation (15% reduction, p<0.03; 19% reduction, p<0.03; and 15% reduction, p<0.01 at 24, 48 and 72 hours, respectively) (Fig 1A). There was significant increase in Caspase 3/7 activity (10.2% increase, p<0.02; 9.7% increase, p<0.05 at 24 and 48 hours, respectively) (Fig 1B). The wounded cells dramatically healed slower than those of control in DLEU1 transient overexpressed 293 cells compared to mock control 293 cells (Fig 2). There were significant increase in Caspase 3/7 activity and significant decrease in cell proliferation in RTX (20ug/ml) treated transient DLEU1 overexpressed 2R (13% increase, p<0.05 and 5% reduction, p<0.02, respectively) compared to RTX treated transient DLEU1 overexpressed Raji. RTX treated transient DLEU1 overexpressed 2R showed significant decrease of expression anti-apoptotic genes, Bcl-xL and Bcl-2 mRNA (<15% reduction, p<0.04 and <20% reduction, p<0.02, respectively). [Display omitted] [Display omitted] ConclusionThese results suggest that 1) overexpression of DLEU1 significantly inhibited cell proliferation, wound healing and induced apoptosis, and 2) we hypothesize that overexpression of DLEU1 in PBL may result in sensitizing BL to chemotherapy induced apoptosis and may result in a potential future therapeutic strategy. Disclosures:No relevant conflicts of interest to declare.