Loucy Cells Research Articles

Abstract 2902: Obinutuzumab (GA101) significantly increases overall survival 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

Abstract Background: Patients who relapse with CD20+ B-NHL and B cell Acute lymphoblastic leukemia (B-LL) have a dismal prognosis, which is 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-LL 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 type II glycoengineered CD20 antibody, mediates enhanced cell death and ADCC vs. RTX (Bologna L et al. JI, 2012), and was recently approved by FDA 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 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. The lentiviral construct, pSico PolII-eGFP-Luc2, was transfected into Raji, Raji 4RH (RTX resistant), U698M and Loucy. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), mice were bred in house under pathogen free conditions, NSG mice were divided into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab 10 mg/kg (generously supplied by Christian Klein, PhD (Roche)), obinutuzumab (30 mg/kg), and rituximab (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 closely monitored for tumor burden and survival for up to 12 weeks ( approx. 80 days) via bioluminescent imaging (BLI) using the IVIS Spectrum system. Results: We demonstrated that 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=0.0002), Raji4RH (p=0.01) and U698-M (p=0.001), respectively. Conclusion: These preliminary studies 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 equal dose of RTX. Citation Format: Aradhana A. Tiwari, Janet Ayello, Carmella van de Ven, Matthew J. Barth, Mitchell S. Cairo. Obinutuzumab (GA101) significantly increases overall survival 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. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2014-2902

Characterization of the Loucy cell line as a model for human peripheral γδ T cells (638.5)

As unique innate‐like lymphocytes, γδ T cells make up a small proportion of circulating T cells. Up to now, there is no widely accepted cell line model for human γδ T cell. Our aim was to investigate whether Loucy cell line shares similarities with γδ T cells derived from human peripheral blood mononuclear cells (PBMC). First, 95% of Loucy cells express the unique γδ T cell receptor (TCR). Second, most Loucy cells were double negative for CD4 and CD8, like most peripheral γδ T cells. Third, Loucy cells express TLR2, like peripheral γδ T cells. At the functional level, Loucy cells proliferated in response to isopentenyl pyrophosphate, a known phosphoantigen for γδ T cell proliferation. Activated Loucy cells express CD69 on cell surface, similar to γδ T cells in activated mixed PBMC cultures. Unlike peripheral γδ T cells, Loucy cell did not express NKG2D. TCR Vδ gene usage in Loucy cells is mainly Vδ3 and to a lesser extent Vδ1, while Vδ2Vγ9 T cells dominate in blood. In addition, 95% of Loucy cells were CD45RO+ (memory), whereas γδ T cells in PBMC were 45% CD45RO+ and 52% CD45RA+ (naive). The only cytokine detected from PHA‐L‐stimulated Loucy cells was TGF‐β, whereas peripheral γδ T cells produce many more cytokines. These results suggest that Loucy cells have many characteristics similar to γδ T cells in PBMC, and this cell line, for some aspects of γδ T cell function, may be useful as an in vitro model for human γδ T cells.Grant Funding Source: Funded in part by UF AES

ABT-737 Restores Prednisolone Sensitivity In Early T-Cell Precursor Acute Lymphoblastic Leukemia With High MEF2C Expression

BackgroundEarly T-cell precursor-acute lymphoblastic leukemia (ETP-ALL) has been identified as high-risk subtype of pediatric T cell acute lymphoblastic leukemia (T-ALL). ETP-ALL is characterized by lack of expression of CD1a and CD8, weak or absent expression of CD5 and aberrant expression of myeloid and hematopoietic stem cell markers, such as CD13, CD33, CD34 and CD117. Because conventional chemotherapy is not fully effective for this subtype, potential therapeutic target should be explored. Here, we assessed gene expression pattern of the transcriptional factors related to differentiation of lymphoid and myeloid cells in ETP-ALL to determine the potential therapeutic target for this particular subtype. Patients and MethodsThirty-one patients with T-ALL treated in Kyoto Prefectural University of Medicine and affiliated hospitals were classified in ETP-ALL or non- ETP ALL based on the results of surface marker analysis by FACS. To compare the gene expression pattern of transcriptional factors related to differentiation of myeloid and lymphoid cells in ETP-ALL with those in non-ETP-ALL, real time RT-PCR (RQ-PCR) was performed to determine expression level of C/EBPa, ID2, NOTCH1, LYL1, IL7R, LMO2, MEF2C, PU.1, and FLT3 in primary leukemic cells from twenty-one patients with T-ALL (ETP-ALL: 9, non ETP-ALL: 12) based on the availability of appropriate RNA. To assess the relationship between high MEF2C expression and prednisolone (PSL) /Bcl2 inhibitor (ABT-737) sensitivity, cell proliferation of Jurkat (not expressing MEF2C) and Loucy (expressing MEF2C) cells cultured with serial concentration of PSL and/or ABT-737 was measured with the WST-1 assay to determine the 50% growth inhibitory concentration (IC50) of these agents. BaF3 cells expressing MEF2C (BaF3-MEF2C) was also generated using retrovirus gene transduction system (MSCV) and cultured with serial concentration of PSL/ABT-737 to determine the IC50 of PSL/ABT-737. Apoptosis was determined by Annexin V/PI staining. Viability of primary leukemic blasts of ETP-ALL and non-ETP-ALL, which were co-cultured with murine stromal cell(MS-5 cell) and treated with PSL and/or ABT-737, was also determined by Annexin V/PI staining. ResultsThirty-one patients diagnosed with T-ALL were classified in ETP-ALL (N=10) or non- ETP ALL (N=21). Although 5 patients in ETP-ALL experienced induction failure (IF), none of the patients in non-ETP-ALL experienced IF (p<0.05). The 5-year EFS in ETP-ALL was significantly inferior than that in non-ETP-ALL ( 40±15.5% vs 76.1±9.2%, log-rank p<0.05). However, the 5-year OS was not significantly different in two groups (67±15.7% in ETP-ALL vs 81±8.5% in non-ETP ALL). RQ-PCR analysis determined that MEF2C and FLT3 was expressed more in ETP-ALL than in non-ETP ALL (MEF2C: p=0.039, FLT3: p=0.014). Because MEF2C augments Bcl2 activity to inhibit the apoptosis, we explored whether high expression of MEF2C was associated with PSL poor response and Bcl2 inhibitor restored the sensitivity to PSL. Combination treatment of PSL and ABT-737 resulted in the significant reduction of IC50 of PSL in Loucy cell (combination index, CI: 0.39). In addition, apoptotic cells increased when Loucy cells were treated with PSL (25mM) in combination with ABT-737 (10nM) compared to be treated with PSL (25mM) alone. In contrast, combination treatment did not reduce significantly IC50 of PSL in Jurkat cell (CI: 1.12). The combination treatment of PSL and ABT-737 also resulted in the significant reduction of IC50 of PSL in BaF3-MEF2C compared to BaF3 cells. Finally, treatment of ABT-737(10nM) in combination with PSL (50 or 200 mM) reduced viability of primary leukemic blasts of ETP-ALL with high expression of MEF2C more profoundly than the treatment of PSL (50 or 200 mM) alone. ConclusionsThese findings suggest that high expression of MEF2C is associated with PSL poor response and Bcl2 inhibitor restores PSL sensitivity in ETP-ALL in vitro. Thus, Bcl2 inhibitor might be therapeutic option for ETP-ALL with high expression of MEF2C. Disclosures:No relevant conflicts of interest to declare.

Obinutuzumab (GA101) Significantly Improves Survival In CD20 Positive Pre-B Cell Lymphoblastic Leukemia (pre-B-ALL) Xenograft Models Compared To Rituximab (RTX): Potential Targeted Therapy In Patients With High Risk Pre-B-ALL

BackgroundObinutuzumab (GA101) is a type II, glycoengineered, humanized anti-CD20 monoclonal antibody (Ab). Obinutuzumab binds with high affinity and selectivity to a type II binding region of the CD20 protein present on all B cells except stem or plasma cells. Unlike type I anti-CD20 monoclonal Abs, type II anti-CD20 Abs promote direct cell death induction without the need for a cross-linking Ab. Patients who relapse with CD20+ B-NHL and early relapsed pre-B-ALL have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012, Barth/Cairo et al. BJH, 2013) and require alternative therapeutic strategies. RTX in combination with FAB/LMB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 95% 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+ NHL/Pre-B-ALL to an increased risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2013). ObjectiveTo evaluate the anti-tumor activity of obinutuzumab vs RTX against pre-B-LL in-vivo in severe combined immune-deficient IL2 receptor gamma chain knockout (NSG) and NOD/SCID mice. MethodsU698-M (Pre-B cell ALL, CD20+, DSMZ, Germany), Loucy cells (T-ALL, CD20- ATCC, Manasass, VA) were maintained in RPMI with 10% FBS at 370C, 5% CO2. The mammalian expression construct ffLUCZeo-pcDNA (kindly provided by L.Cooper, MD,PhD) was transfected into U698M and Loucy (Luc+) tumor targets for later evaluation by bioluminescent imaging (BLI). Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, no functional T, B and NK cells) and NOD/SCID (absence of functional T and B cells) mice were bred in-house and treated in pathogen free conditions at the animal facility. The in vivo cytotoxic activity of obinutuzumab and RTX was determined by dividing NSG and NOD/SCID mice into 5 groups: PBS only (control), isotype control (IgG), obinutuzumab (10 mg/kg) (kindly provided by C. Klein, Roche Glycart AG), obinutuzumab (30 mg/kg), and rituximab (30 mg/kg). Mice received intravenous injections of Luc+ U698M and Loucy cells at 5x106 tumor cells/mouse. 6-8 days after tumor cell injection, mice were injected every 7 days with the respective therapy for 8 weeks. Mice were closely monitored for tumor burden progression/regression and survival for up to 12 weeks (approx. 80 days) via BLI using the IVIS Spectrum System. ResultsWe observed that obinutuzumab was significantly more effective than RTX when administered at the same doses in established pre-B-ALL xenografts. In the NSG mouse model, at 30 days tumor volume in mice receiving 30 mg/kg of obinutuzumab was significantly decreased when compared to mice receiving 10 mg/kg obinutuzumab or 30 mg/kg of RTX (p<0.0001, p<0.002 respectively). At 11 weeks, mice receiving 30 mg/kg of obinutuzumab showed no increase in tumor volume compared to week 4. The overall survival following obinutuzumab at 30 mg/kg was 80 days compared to 48 days by obinutuzumab at 10mg/kg (p=0.001) and 30 days following RTX treatment at 30mg/kg)(p=0.002) (Figure-1A). Relative tumor luminescence activity on day 8 was significantly decreased in mice receiving 30 mg/kg of obinutuzumab (8x105 RLU) compared to 10 mg/kg of obinutuzumab (7.7x105 RLU) or 30 mg/kg of RTX (9.9x105 RLU) compared to IgG isotype (2.0x106 RLU, p=0.02,p=0.07,p=0.1, respectively).Similarly, in the NOD/SCID mouse model, at day 80, we observed a significant decrease in tumor luminescence when mice were treated with 30 mg/kg obinutuzumab compared to 10 mg/kg obinutuzumab or 30 mg/kg rituximab (p=0.001). Furthermore, at day 80 there was 100% survival in animals with 30 mg/kg obinutuzumab compared to 60% following rituximab at 30 mg/kg ( p=0.001) or 40% obinutuzumab (10mg/kg; p=0.001) (Figure1B). [Display omitted] ConclusionThese preliminary studies in two groups of pre-B-ALL xenograft mice (NSG and NOD/SCID), demonstrated that the antitumor effect of obinutuzumab at 30 mg/kg significantly increases survival and decreases the tumor burden in Pre-B-ALL xenografts compared to equal dose of RTX. Further studies are underway to determine the mechanism(s) responsible for the significantly increased efficacy of obinutuzumab vs. RTX in pre-B-ALL xenografts. Disclosures:Cairo:Roche/Genentech: advisory board Other.

Obinutuzumab (GA101) Significantly Enhances Cell Death and ADCC Compared to Rituximab Against CD20+ sensitive and Rituximab Resistant B-Cell Non-Hodgkin Lymphoma (NHL) and Lymphoblastic Leukemia (BLL)

AbstractAbstract 4865 Background:Patients who relapse with CD20+ B-NHL and B cell lymphoblastic leukemia (B-LL) have a dismal prognosis, often associated with chemotherapy resistance (Cairo et al. JCO, 2012,Mils/Cairo et al. BJH,2012) and often require alternative therapeutic strategies. Rituximab (RTX) in combination with FAB 96 chemotherapy is a safe, well-tolerated treatment that is associated with > 90% EFS in children with newly diagnosed and advanced mature B-Cell NHL (Cairo M.S. et al. ASCO, 2010). Resistance to RTX, however, may predispose patients with CD20+ NHL to an increase risk of relapse and or disease progression (Barth/Cairo et al. BJH, 2012; Tsai et al. Cl. Can. Res, 2012). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, mediates enhanced cell death vs RTX and has a glycoengineered Fc region that induces significantly enhanced ADCC (Mössner et al. Bld, 2010; Niederfellner G. et al. Bld, 2011; Bologna L et al. JI, 2012). Objective:To evaluate the in-vitro efficacy of GA101 compared to RTX against RTX sensitive and resistant CD20+ B-NHL and B-LL cell lines. Methods:Raji (CD20+,ATCC, Manhass, VA), U698-M (CD20+, DSMZ, Germany), Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) and Raji-2R and Raji-4RH (generously supplied by M. Barth, Roswell Park Cancer Institute) were cultured in RPMI with 10% FBS and incubated with GA101 and/or RTX at 100 μg/ml for 24 hrs (n=6), 48 and 72 hrs (n=5). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. Loucy cells (CD20−) were used as the negative control. The caspase 3/7 activity was measured by FAM caspase 3/7 assay kit by FLICA™ methodology. RSCL, RRCL, U698-M and Loucy were incubated with GA101 and RTX treatment for 24, 48 and 72 hrs, and caspase3/7 activity was detected by FACS using 488 nm excitation and emission filter (n=3). ADCC were performed with K562-IL-15–41BBL expanded NK cells (Ayello/Cairo et al. ASH, 2010) as well as IL-2 expanded NK cells, at 20:1 effector: target ratio (E: T, n=3) using europium release assay (Perkin-Elmer). Results:GA101 induced significantly more cell death compared to RTX in B-NHL and BLL cell lines. (Table-1)24 hrs48 hrsGroupsRTX ObinutuzumabP-valueRTX ObinutuzumabP-valueLoucy CD20−5.32±2.5%6.15±2.8%5.8±1.8%6.8±1.3%U698-M CD20+26.36±2.6%40.5±2.01%(p<.0001)25.1±2.0%.47.38±4.9%(p<.0001)Raji CD20+32.7±6.8%45.1±3.3%(p<.005)25.1±2.0%.35.6±9.1%(p<.001)Raji2R CD20+ Resistant2.9±1.1%17.6±1.3%(p<.001)7.8±2.45%18.2±0.09%(p<.001)Raji4RH CD20+ Resistant2.1±1.5%15.8±2.2%(p<.001)7.9±1.3%19.7±2.2%(p<.001)GA101 vs RTX shows a significantly increase in caspase 3/7 activity in Raji 16.92±0.84% vs 11.76±0.08% compared to Raji2R 6.7±0.62% vs 2.8±0.7%, Raji4RH 5.8±0.35% vs 2.0±0.3% and U698-M 12.54±0.44% vs 9.6±0.95% compared to Loucy 3.22±0.45% vs 2.59±0.05%, respectively, at 24 hrs of treatment (p<0.0001). GA101 vs RTX also elicited a significant increase a ADCC with K562-IL15–41BBL expanded NK cells, in Raji 73.8±8.1% vs 56.81±4.6% compared to Raji-2R 38.0±2.0% vs 21.6±1.2%, Raji-4RH 40.0±1.6% vs 0.5±1.1% and U698-M 70.0±1.6% vs 45.56±0.1%, compared to Loucy 21.67±0.48% vs 15.92±0.52%, respectively (p<0.001) at day 7.The IL-2 alone expanded Hu-NK cells demonstrated a reduction of 10–20% cytotoxicity compared to K562-IL15–41BBL Hu-NK cells at day 7 against BLL, RSCL and RRCL, in-vitro. Conclusion:Obinutuzumab compared to RTX significantly enhanced cell death, caspase3/7 activity and NK mediated ADCC in sensitive and RTX resistant B-NHL and B-LL. Obinutuzumab represents a promising candidate for treating RTX sensitive and resistant CD20+ B-Cell Lymphomas and lymphoblastic leukemia. Further studies will investigate the combination of activated NK cells or chemotherapy that may enhance or synergize with the efficacy of GA101 (Obinutuzumab) both in -vitro and in-vivo in xenografted NOD/SCID mice. Disclosures:No relevant conflicts of interest to declare.

Effect of obinutuzumab (GA101), a type II glycoengineered monoclonal antibody targeting CD20, against rituximab resistant and sensitive cell lines in B-cell non-Hodgkin lymphoma.

e21150 Background: : Relapse or insufficient response or resistance to Rituximab (RTX) has evolved as a challenge in therapy of B-cell NHL. RTX in combination with FAB chemotherapy is a safe, well tolerated and associated with &gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). Obinutuzumab (GA101), a novel type II glyco-engineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glycoengineered Fc region has shown to cause significantly enhanced ADCC (Mössner et al, Bld 2010; Niederfellner G. et al, Bld 2011). We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines(RRCL), Raji-2R and Raji-4RH, respectively (generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY. Barth M. et al, ASH 2010). Methods: All cell lines were cultured in RPMI with 10% FBS and incubated with escalating doses of GA101 (1-1000 µg/ml) for 24 hrs (n=10), 48 and 72 hrs (n=6). Cell death was evaluated by staining with AnnexinV/7AAD and flow-cytometry. B-Cell Lymphoblastic Lymphoma (BLL) U-698-M cells (CD20+) were used as the positive and Loucy cells (CD20-) (T-ALL) were used as the negative controls. Results: At 48 hrs 100 µg/ml of GA101, Raji and U-698-M demonstrated highest cell death of 33.62±5.7% and 37.8± 3.9%, respectively; while Raji-2R and Raji-4RH cell death was 22.8±1.8% and 10.31±0.81% (p&lt;0.02) and (p&lt;0.001) respectively. At 1000 µg/ml of GA101 at 24 hrs, Raji demonstrated increased cell death 40.27±9.1% compared with Raji2R 14.1±0.07 and Raji4RH 20.3± 3.2 (p&lt;0.001) and (p&lt;0.001) respectively. Conclusions: GA101 represents a promising candidate for treating RTX resistant B-Cell Lymphomas and related CD20+B-Cell malignancies. Further studies should aim to identify novel approaches such as combination with activated NK Cells or chemotherapeutic agents that may enhance or synergize with the efficacy of GA101. Further investigations aim to elucidate the mechanisms responsible for minimal cell death induction on the U698M and Raji cell lines.

Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma

Abstract Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with &amp;gt; 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p&amp;lt;0.05) and Raji vs Raji-4RH (p&amp;lt;0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 516. doi:1538-7445.AM2012-516

Activation of Paired-homeobox gene PITX1 by del(5)(q31) in T-cell acute lymphoblastic leukemia

In T-cell acute lymphoblasic leukemia (T-ALL), neoplastic chromosomal rearrangements are known to deregulate members of the homeobox gene families NKL and HOXA. Here, analysis of T-ALL cell lines and primary cells identified aberrant expression of a third homeobox gene group, the Paired (PRD) class. LOUCY cells revealed chromosomal deletion at 5q31, which targets the downstream regulatory region of the PRD homeobox gene PITX1, removing a STAT1 binding site. STAT1 mediates repressive interleukin 2 (IL2)–STAT1 signaling, implicating IL2 pathway avoidance as a possible activation mechanism. Among primary T-ALL samples, 2/22 (9%) aberrantly expressed PITX1, highlighting the importance of this gene. Forced expression of PITX1 in JURKAT cells and subsequent target gene analysis prompted deregulation of genes involved in T-cell development including HES1, JUN, NKX3-1, RUNX1, RUNX2, and TRIB2. Taken together, our data show leukemic activation of PITX1, a novice PRD-class homeobox gene in a subset of early-staged T-ALL, which may promote leukemogenesis by inhibiting T-cell development.

Transcriptional deregulation of oncogenic myocyte enhancer factor 2C in T-cell acute lymphoblastic leukemia

Myocyte enhancer factor 2C (MEF2C) encodes a transcription factor which is ectopically expressed in T-cell acute lymphoblastic leukemia (T-ALL) cell lines, deregulated directly by ectopically expressed homeodomain protein NKX2-5 or by loss of promoter regions via del(5)(q14). Here, we analyzed the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix proteins, STAT5, and HOXA9/HOXA10. Chromatin immunoprecipitation and overexpression analyses demonstrated direct activation by GFI1B and LYL1 and inhibition by STAT5. HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of regulation via NMYC interactor (NMI) and STAT5. Lacking comma: Chromosomal deletion of the STAT5 binding site in LOUCY cells reduced protein levels of STAT5 in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5 signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that the expression of MEF2C in T-ALL cells is principally deregulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory developmental STAT5 signaling.

Transcriptional Regulation of Oncogenic MEF2C In T-Cell Acute Lymphoblastic Leukemia (T-ALL).

AbstractAbstract 3636Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS-box family which is physiologically expressed in hematopoietic stem cells and during development of B-cells. Recently, we identified ectopic expression of MEF2C in T-cell acute lymphoblastic leukemia (T-ALL) cell lines activated either via chromosomally mediated ectopic expression of homeodomain protein NKX2-5 or via deletion of non-coding exon and promoter regions at 5q14, suggesting loss of negative regulatory elements. Our aim was to identify additional transcriptional regulators of MEF2C in T-ALL. Therefore, we analyzed the sequence of the MEF2C 5′-region, thus identifying potential regulatory binding sites for GFI1B, basic helix-loop-helix (bHLH) proteins, STAT5 and HOXA9/HOXA10. Overexpression studies demonstrated MEF2C activation by GFI1B (strong), LYL1 and TAL1 leukemic bHLH proteins (weak), and inhibition by STAT5 (strong) and HOXA9/HOXA10 (weak). Chromatin-Immuno-Precipitation analysis demonstrated direct binding of GFI1B, LYL1 and STAT5 but not of HOXA10 to the MEF2C 5′-region in T-ALL cells. However, HOXA9/HOXA10 activated expression of NMYC which in turn mediated MEF2C repression, indicating an indirect mode of MEF2C regulation. Chromosomal deletion of the 5′-MEF2C STAT5 binding site in LOUCY cells by del(5)(q14), reduced expression levels of STAT5 protein in some MEF2C-positve T-ALL cell lines, and the presence of inhibitory IL7-JAK-STAT5-signaling highlighted the repressive impact of this factor in MEF2C regulation. Taken together, our results indicate that ectopic expression of MEF2C in T-ALL cells is mainly regulated via activating leukemic transcription factors GFI1B or NKX2-5 and by escaping inhibitory STAT5-signaling. Disclosures:No relevant conflicts of interest to declare.

SET-NUP214 fusion in acute myeloid leukemia- and T-cell acute lymphoblastic leukemia-derived cell lines

BackgroundSET-NUP214 fusion resulting from a recurrent cryptic deletion, del(9)(q34.11q34.13) has recently been described in T-cell acute lymphoblastic leukemia (T-ALL) and in one case of acute myeloid leukemia (AML). The fusion protein appears to promote elevated expression of HOXA cluster genes in T-ALL and may contribute to the pathogenesis of the disease. We screened a panel of ALL and AML cell lines for SET-NUP214 expression to find model systems that might help to elucidate the cellular function of this fusion gene.ResultsOf 141 human leukemia/lymphoma cell lines tested, only the T-ALL cell line LOUCY and the AML cell line MEGAL expressed the SET(TAF-Iβ)-NUP214 fusion gene transcript. RT-PCR analysis specifically recognizing the alternative first exons of the two TAF-I isoforms revealed that the cell lines also expressed TAF-Iα-NUP214 mRNA. Results of fluorescence in situ hybridization (FISH) and array-based copy number analysis were both consistent with del(9)(q34.11q34.13) as described. Quantitative genomic PCR also confirmed loss of genomic material between SET and NUP214 in both cell lines. Genomic sequencing localized the breakpoints of the SET gene to regions downstream of the stop codon and to NUP214 intron 17/18 in both LOUCY and MEGAL cells. Both cell lines expressed the 140 kDa SET-NUP214 fusion protein.ConclusionCell lines LOUCY and MEGAL express the recently described SET-NUP214 fusion gene. Of special note is that the formation of the SET exon 7/NUP214 exon 18 gene transcript requires alternative splicing as the SET breakpoint is located downstream of the stop codon in exon 8. The cell lines are promising model systems for SET-NUP214 studies and should facilitate investigating cellular functions of the the SET-NUP214 protein.

Aberrant Expression and Activation of Nuclear Factor of Activated T cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL) May Contribute to Leukemogenesis

Nuclear Factor of Activated T-cells 1 (NFAT1) is a member of the NFAT family of transcription factors (NFAT1-NFAT5) that has been shown to play an important role in regulating genes related to T-cell expansion, differentiation, and apoptosis. As murine NFAT1 knockout mice exhibit lymphoproliferative disease, we hypothesize that aberrant expression of NFAT1 may impact cell cycle dysregulation underlying T-ALL pathogenesis. Four T-ALL cell lines – including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, -2, -18, del(2) (p21p23), del(18) (p11.2)), Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line MOLT-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA), established from peripheral blood of T-ALL patients were analyzed and compared to normal resting adult CD4+ T-cells. Flow cytometry analysis was performed as previously described including CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 to determine maturation stage; MOLT-4 being the most primitive and Jurkat the most mature.Methods: Growth curves were determined and proliferation potential was determined using MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay. The cell cycle phase distribution was assessed by flow cytometry analysis of Hoechst 33342-stained cells. mRNA expression was examined by quantitative rtPCR using TaqMan probes (ABI) on cDNA derived from TRIzol purified total RNA. For stimulation, we used 2 μM ionomycin treatment for 3 hours. Proteins were prepared as whole cell extract from radioimmunoprecipitation assay (RIPA) buffer lysed cells. Protein expression was evaluated by Western blotting analysis of 20 μg of proteins using anti-NFAT1 antibody (BD Biosciences) and anti-γ-tubulin antibody (Sigma) as a loading control. Transient transfection of GFP vector or plasmid containing the wild-type or constitutively active NFAT1 gene along with the GFP gene was performed by either electroporation (Amaxa) or Lipofectamine 2000 (Invitrogen). GFP positive cells were sorted by FACSAria sorter.Results: Despite their different maturation states, all cell lines (except Loucy) have similar high growth rates. However, the cell cycle distribution analysis of Hoechst-stained cells revealed a lower proportion of Loucy cells in the G0/G1 phase (35% vs. 48–51% for the other 3 cell lines) and a higher proportion in the G2/M phase (37% vs. 19–24% for the other 3 cell lines). In addition, Loucy cells have a higher rate of apoptosis as measured by Annexin V staining. Analysis of NFAT1 expression demonstrated decreased levels of NFAT1 mRNA (30-70-fold) and protein (2-10-fold) in these cell lines compared to resting adult peripheral blood CD4+ T lymphocytes. [Display omitted] Moreover, ionomycin stimulation of the calcium-calcineurin pathway in these cells revealed aberrant activation of NFAT1. There was no dephosphorylated or activated NFAT1 in MOLT-4 and Jurkat cells; in contrast, dephosphorylated or activated NFAT1 was degraded in CCRF-CEM and Loucy. As NFAT1 is implicated in the regulation of cell cycle and apoptosis, the expression of cell cycle and apoptosis genes was measured by qrtPCR. Consistent with the negative regulatory role of NFAT1 in cell cycle, T-ALL cells expressing low level of NFAT1 showed upregulation of cyclin A2 (20-80-fold), cyclin E2 (5-8-fold) and CDK4 (3-7-fold) as well as downregulation of p21Cip1 (20-470-fold) and p27Kip1 (20-180-fold). In addition, these cells also demonstrated downregulation of the expression of the pro-apoptotic gene Nur77 (2.5-10-fold). Introduction of exogenous NFAT1 gene into Jurkat cells resulted in decreased proliferation rate to 64% and 42%, for wild-type and constitutively active form of NFAT1 gene, respectively, compare to the cells transfected with the empty GFP vector.Conclusion: Aberrant expression of NFAT1 contributes to leukemogenesis via dysregulation of proliferation and apoptosis. Targeted NFAT1 expression may be an effective therapeutic strategy in T-ALL.

Targeting Histone Deacetylase 6 and the Aggresome Pathway in Acute Lymphoblastic Leukemia Cells.

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Despite effective chemotherapy, 20% of patients will relapse. Therefore, it is critical that we identify novel therapies to treat ALL. We are studying a new small molecule compound known as tubacin (tubulin acetylation inducer) that selectively inhibits histone deacetylase 6 (HDAC6). HDAC6 binds to polyubiquitinated misfolded proteins and to dynein motor proteins, including alpha-tubulin, thereby recruiting misfolded or unwanted proteins to aggresomes and subsequent degradation by the lysosome. Tubacin was discovered through a chemical genetic screen of a 7,392 small molecule library and was found to induce acetylation of alpha-tubulin by inhibiting one of the two catalytic domains of HDAC6. This inhibition disrupts the interaction of HDAC6 with dynein resulting in marked accumulation of ubiquitinated proteins. Previous work demonstrated that treatment of multiple myeloma cells with tubacin inhibited growth at low micromolar concentrations. Tubacin does not appear to affect global histone acetylation, gene expression, or cell cycle regulation. To determine the effects of tubacin in human ALL cells, we treated both B- and T-cell ALL cell lines with varying concentrations of the drug and performed MTT assays. In T-ALL cells (Jurkat, Loucy), the IC50 of tubacin was found to be 1 to 3 uM, while in B-cell ALL cells (REH, Nalm-6), the IC50 was 2 to 5uM. Tubacin induced apoptosis of Jurkat and Loucy cells stained with Annexin V and propidium iodide. Within 12 hours, we observed increased protein polyubiquitination and PARP cleavage, but no difference in Rb phosphorylation in cells treated with tubacin. Furthermore, tubacin treatment increased acetylation of alpha-tubulin in Loucy and Jurkat cells within 3 hours. To study whether tubacin was toxic to normal hematopoietic cells, we treated human bone marrow cells cultured in methylcellulose containing IL-3, IL-6, and Stem Cell Factor with varying concentrations of tubacin. The IC50 of 20uM was determined from numbers of colonies plated in triplicate. Similarly, treatment of normal human lymphocytes cultured in IL-2, demonstrated an IC50 of 16uM. Finally, we examined the effects of tubacin on growth of primary ALL cells. Bone marrow cells from three patients with B-cell ALL at diagnosis were cultured in tubacin at varying concentrations and MTT assays were performed. In two of the three ALL samples, the IC50 was less than 5uM. Experiments to study the effects of tubacin in mouse models of leukemia are in progress. Our results suggest that inhibition of HDAC6 and the aggresome pathway provides a novel approach to treat ALL.

Dysregulation of Nuclear Factor of Activated T-Cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL): Possible Role of Inactivated NFAT1 in Leukemogenesis.

Introduction: NFAT1 is a transcription factor integral for the regulation of T-cell proliferation, differentiation, and apoptosis. NFAT1 is present in the cytoplasm of resting T-cells and upon stimulation (ionomycin, antigen or anti-CD3) is dephosphorylated via calcineurin and translocates to the nucleus to associate with other transcription factors (AP1). We sought to determine whether NFAT1 regulation in T-cells contributes to leukemogenesis. Initial in vitro analyses incorporated several T-ALL cell lines all derived from the peripheral blood of patients including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, −2, −18, del(2) (p21p23), del(18) (p11.2)) Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line Molt-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA).Methods: T-ALL cell lines were cultured in accordance with ATCC guidelines. Surface marker analysis for CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 was performed (BD Biosciences). Cytoplasmic and nuclear extracts were prepared from cell lines and control adult blood (AB) CD3+ cells. Cell lysates (20μg) were examined by Western blot. Blots were probed with anti-NFAT1 antibody (BD Biosciences) and protein loading controls β2-microglobulin (Abcam) and lamin A/C (Santa Cruz Biotechnology). Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce) and quantitated using NIH imageJ software. Apoptotic assays were performed by treating samples with 0.832 μM cyclosporin A (CsA) for 48 hrs, then determining the percentage of early apoptotic cells using an Annexin V/Propidium iodide flow cytometric kit (Calbiochem).Results: Surface marker analysis confirmed that Loucy, Jurkat, and CCRF-CEM cells possess a mature phenotype, whereas Molt-4 cells possess a more immature phenotype (Table 1). Western blot analysis showed Loucy cells were unique in that NFAT1 was absent in the nuclear fraction, indicating NFAT1 is only present in it's inactive form in this cell line. Among the T-ALL cell lines, only Loucy showed no difference in the proportion of apoptotic cells following CsA treatment.Table 1Phenotype Analysis of T-ALL Cell LinesSurface AntigensCCRF-CEMJURKATLOUCYMOLT-4CD283.9796.164.9691.53CD799.7487.8499.9657.88CD318.2252.1596.488.33CD462.4254.5612.6242.64CD897.425.320.8183.19CD3414.802.620.6555.78CD3893.5989.4092.9299.66HLA-DR2.312.620.560.33Percentage of Total CellsConclusions: Despite Loucy cells' mature phenotype, they exhibited distinct NFAT1 dysregulation compared to other mature cell lines. CsA treatment of Loucy cells failed to effect NFAT1 translocation to the nucleus and induction of apoptosis, as NFAT1 remained in the cytoplasm. Loucy's insensitivity to CsA with regards to apoptosis signaling supports the idea for a role for NFAT1 in Ca(2+) signaling cascade for apoptosis in T-cells. These results also suggest a possible role of cytosolic NFAT1 in leukemiogenesis, since the amount of protein seen in whole cell extracts between the various cell lines was nearly the same. The translocation in Loucy cells is in the same chromosomal location as the NFAT1 gene locus. Studies are ongoing to understand the role of NFAT1 in T-ALL initiation and progression.

Anti-proliferative activity of CC-5013 in 5q- myelodysplastic syndrome (MDS) and acute lymphocytic leukemia (ALL) cell lines

6618 Background: Phase I/II studies of CC-5013 (Revimid™) in MDS have shown erythropoietic and cytogenetic responses, especially among 5q- patients. The 5q deletion is a primary chromosomal change among various hematological malignancies including MDS, and it affects expression of genes on the locus including the tumor suppressor interferon regulatory factor- 1 (IRF-1). The activity of CC-5013 was tested in various 5q mutant MDS, AML, and ALL cell lines to assess associations between CC-5013 efficacy and genetic alterations. Methods: The MDS cell line MUTZ-1, AML cell lines KG-1 and UT-7, and ALL cell line Loucy, along with control cell lines Jurkat and MUTZ-5 were tested for sensitivity to CC-5013 using 3H-thymidine incorporation, annexinV and propidium iodide (PI) staining. IRF-1 expression and CD19 or EPO receptor-induced Akt phosphorylation were analyzed by western blot. Gene expression was analyzed using the Affymetrix U133 GeneChip. Results: The cell proliferation IC50 values for MUTZ-1, KG-1, and Loucy cells are 2.5, 66 and 100 μM respectively. The MUTZ-5, UT-7 and Jurkat cell line IC50s were well above 100 μM. Cell cycle analysis indicates CC-5013 causes G0/G1 arrest in the sensitive cell lines. In MUTZ-1 cells, there is a 3–8% increase in apoptosis with CC-5013. MUTZ-1 and KG-1 cells express less IRF-1 protein than UT-7 cells, consistent with their 5q status. CC-5013 inhibited Akt phosphosphorylation in response to CD19 or EPO stimulation of MUTZ-1 cells. Genes regulated by CC-5013 in MUTZ-1 cells include genes involved in cell adhesion and a metalloprotease. Conclusions: CC-5013 has preferential anti-proliferative activity against 5q mutant cell lines. This inhibition appears to be due to G0/G1 arrest with a small apoptotic component, and correlates with IRF-1 expression. In MUTZ-1 cells, CC-5013 interferes with growth signals via Akt and increases expression of genes found at the 5q locus. These molecular events may explain the sensitivity of 5q mutant hematopoietic tumor clones to CC-5013-induced toxicity. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celgene Corp. Celgene Corp. Celgene Corp.