Obatoclax (GX15-070) Overcomes Glucocorticoid-Resistance In Acute Lymphoblastic Leukemia through Induction of Apoptosis and Autophagy

Abstract Abstract 1809 Glucocorticoids (GC) are common components in many chemotherapeutic protocols for lymphoid/myeloid malignancies, including acute lymphoblastic leukemia (ALL). However, patients often develop resistance to GC on relapse. Resistance to GC in ALL can be associated with defects in apoptosis machinery, but not in the GC receptor. Thus, targeting downstream molecules may lead to the development of new therapeutic strategies. GC-induced apoptosis is through the intrinsic mitochondria-dependent pathway. The BCL-2 family proteins are central regulatory proteins in this pathway. We hypothesized that targeting anti-apoptotic MCL-1 might be effective among the BCL-2 family proteins, since (1) we recognized that treatment with dexamethasone (Dex) in CCRF-CEM or Molt-4 T-ALL cells slightly induce MCL-1 and the expression level of MCL-1 is higher in Dex-resistant ALL cells compared with that in Dex-sensitive cells; (2) recent studies have demonstrated that increased expression of MCL-1 associates with GC resistance. In support of our hypothesis, down-regulation of MCL-1 by shRNA enhances Dex-induced cell death. We then pharmacologically inactivate MCL-1 function by GX15-070 (obatoclax), a BH3 mimetic small molecule that targets anti-apoptotic BCL-2 family proteins including BCL-2, BCL-XL, and MCL-1. Treatment with GX15-070 in both Dex-sensitive and -resistant ALL cells shows effective growth inhibition and cell death. GX15-070 induces caspase-3 cleavage and increases Annexin V-positive population, indicative of apoptosis. Before the onset of apoptosis, GX15-070 induces LC3 conversion as well as p62 degradation, both of which are autophagic cell death markers. A pro-apoptotic molecule BAK is released from BAK/MCL-1 complex following GX15-070 treatment. Consistently, down-regulation of BAK reduces caspase-3 cleavage and cell death, but does not alter LC3 conversion. In contrast, down-regulation of ATG5, an autophagy regulator, decreases LC3 conversion and cell death, but does not alter caspase-3 cleavage, suggesting that apoptosis and autophagy induced by GX15-070 are independently regulated. Down-regulation of Beclin-1, which is capable of crosstalk between apoptosis and autophagy, affects GX15-070-induced cell death through apoptosis but not autophagy. Taken together, GX15-070 treatment in ALL could be an alternative regimen to overcome glucocorticoid resistance by inducing BAK-dependent apoptosis and ATG5-dependent autophagy. Enhanced anti-apoptotic BCL-2 family protein expression has been observed in several types of tumors. Targeting these proteins is therefore an attractive strategy for restoring the apoptosis process in tumor cells. Among the small molecule BCL-2 inhibitors, ABT-737 and its analog ABT-263 are the leading compounds currently in clinical development. However, these molecules have an affinity only with BCL-2 and BCL-XL, but not with MCL-1. Thus, ABT-737 can not be effective as a single agent therapeutic for ALL when MCL-1 is overexpressed. In contrast, GX15-070 can overcome the resistance conferred by high level of MCL-1. Our results suggest that GX15-070 could be useful as a single agent therapeutic against ALL and that the activity/expression of anti-apoptotic proteins could be a biomarker to determine the treatment strategy to ALL patients. (Supported by NIH R01CA134473 and the William Lawrence and Blanche Hughes Foundation) Disclosures: No relevant conflicts of interest to declare.

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JAK2 Aberrations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v128.22.583.583 ◽

2016 ◽

Vol 128 (22) ◽

pp. 583-583

Author(s):

Elisabeth M.P. Steeghs ◽

Isabel S. Jerchel ◽

Willemieke de Goffau-Nobel ◽

Alex Q. Hoogkamer ◽

Judith M. Boer ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Induced Resistance ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Leukemic Cell ◽

Leukemic Cells ◽

Cell Precursor

Abstract Background In high risk pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients, gain of function mutations and translocations affecting JAK2 have been described. These mutations and translocations result in aberrant kinase signaling and may therefore serve as an ideal target for precision medicines. Aim Evaluate the frequency and prognosis of JAK2 lesions among different subtypes of childhood BCP-ALL, and study the efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib. Methods This study comprised 77 BCR-ABL1-like cases and 76 B-other cases which were screened for JAK2 translocations using RT-PCR. Furthermore a representative pediatric cohort of 461 newly diagnosed BCP-ALL cases was screened for JAK2 mutations using targeted next-generation sequencing. Clinical analyses were performed in 341 BCP-ALL patients. Patient-derived-xenograft (PDX) cells were isolated from NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, which were injected with primary leukemic cells. Purity of PDX cells was enriched to over 90% and presence or absence of JAK2 lesions was validated. PDX and primary leukemic cells were exposed to a dilution series of momelotinib or ruxolitinib for four days. Where indicated, cells were pre-incubated with 25 ng/ml TSLP for 1 hour. In mono-culture assays, cytotoxicity was quantified using MTT and in co-culture assays flow cytometry was used. Leukemic cells were discriminated from mesenchymal stromal cells (MSCs) using CD19 and viability was assessed by Annexin V and Propidium Iodide. Western blotting was used to study protein expression levels. Results JAK2 translocations were detected in 6.5% of BCR-ABL1-like cases (3 PAX5-JAK2 cases, 1 TERF2-JAK2 case and 1 BCR-JAK2 case), but not in B-other cases. JAK2 mutations were identified in 3.5% of all BCP-ALL cases, which included JAK2 mutations in BCR-ABL1-like (7.6%), B-other (11.9%), and high hyperdiploid cases (1.6%), but not in MLL rearranged, BCR-ABL1-positive, ETV6-RUNX1-positive or TCF3-PBX1-positive cases. Cumulative incidence of relapse in patients harboring JAK2 lesions was as poor as in JAK2 wildtype BCR-ABL1-like and B-other patients. Efficacy of the JAK1/2 inhibitors momelotinib and ruxolitinib was examined in JAK2 lesion positive (primary and PDX) leukemic cells. Inhibitors were cytotoxic in both translocated and mutated cells, although efficacy in JAK2 mutated cells highly depended on CRLF2 activation by TSLP. CRLF2 activation resulted in downstream STAT5 activation and sensitization towards ruxolitinib compared to unstimulated cells (p < 0.05). Cells harboring JAK2 translocations signaled independently of CRLF2. Although momelotinib and ruxolitinib exposure blocked downstream STAT1/5 phosphorylation, both inhibitors also induced accumulation of phosphorylated JAK2Y1007. Consequently, release of the inhibitors resulted in a profound re-activation of JAK2 signaling, observed by upregulation of downstream STAT1/5 signaling. Furthermore, we observed microenvironment-induced resistance. Culturing leukemic cells in the presence of primary bone marrow MSCs induced resistance to ruxolitinib, compared to leukemic cells in single cultures (p < 0.05). A similar trend was observed for momelotinib. In addition, patients harboring JAK2 mutations displayed a heterogeneous leukemic cell population. Mouse xenograft models revealed different outgrowth patterns of leukemic cells, in which the JAK2 mutated clone persisted, decreased or even disappeared, resulting in outgrowth of JAK2 wildtype leukemic cells. Moreover, JAK2 mutations were not mutually exclusive for other pathway mutations (e.g. KRAS). Conclusion JAK2 translocations and mutations were detected in poor prognostic BCP-ALL cases. In ex vivo assays, the JAK1/2 inhibitors momelotinib and ruxolitinib were cytotoxic in JAK2 aberrant cells. Despite these promising findings, we identified certain limitations of these inhibitors. Inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon their release. Furthermore, our data suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and by microenvironment-induced resistance. Taken together, our data yield important directives for the clinical use of JAK inhibitors in pediatric BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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Blinatumomab + Ponatinib for Relapsed Ph1-Positive Acute Lymphoblastic Leukemia: The French Experience

Blood ◽

10.1182/blood-2018-99-111546 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4014-4014 ◽

Cited By ~ 8

Author(s):

Marie-Anne Couturier ◽

Xavier Thomas ◽

Francoise Huguet ◽

Céline Berthon ◽

Célestine Simand ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Disease Risk ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Philadelphia Chromosome ◽

New Drugs ◽

Molecular Response ◽

Complete Molecular Response

Abstract Prognosis of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph1+ ALL) has been considerably improved since the beginning of the BCR-ABL1 tyrosine kinase inhibitors (TKI) era 20 years ago. However, the prognosis of patients with a refractory/relapsed (including molecular relapse) disease is still very dismal. New drugs or combination of new drugs may improve outcomes of these patients. For example, ponatinib, a 3rd-generation oral TKI, known to have activity against BCR-ABL1 T315I mutations, has shown some efficacy in this context. Similarly, a recent study has reported very encouraging results of the bispecific anti-CD3/CD19 monoclonal antibody blinatumomab as single-agent in refractory/relapsed Ph1+ ALL. Data regarding the efficacy and tolerance of a combination of blinatumomab+ponatinib (blina/pona) are still scarce. This was a retrospective study with the aim to report outcomes of patients receiving a combination of blina/pona for refractory/relapsed Ph1+ ALL in France. Fifteen adults from 8 French centers were identified and data were collected by physicians of each centers, then gathered for the purpose of this study. There were 9 males and 6 females, with a median age of 53 years (range:17-72). All patients, but 2 with blast crisis of chronic myeloid leukemia, had de novo Ph1+ ALL. Four cases had BCR-ABL1 T315I mutation. Patients received the blina/pona combination, either after a first (n=8) or a second (n=7) cytologic relapse. There was no refractory patient in theses series. Previous allograft and autograft has been performed in 7 and 2 patients, respectively. The majority of patients (n=12) had previously received 2 or more lines of TKI. The median time between the first cycle of blina/pona and diagnosis was 14 months (range: 8-40). The median number of blinatumomab cycle (28 mg/day by continuous infusion for 28 days every 6 weeks) administered per patient was 3 (range: 1-6) while ponatinib was concomittantly administered continuously at an initial dose of 45 mg once daily in 11 pts (73%) and 30 mg in 4 pts (27%). Median duration of ponatinib administration was 4 months (range: 1.1-10.9) from first blinatumomab cycle. The toxicity profile was safe: all patients received a complete first cycle without grade 3-4 adverse events. After cycle 1, blinatumomab was stopped in 47% of cases because of neurologic events in 4 and infections in 3; ponatinib was stopped in 33% of cases because of neurologic events in 3, fluid retention in 1 and severe arteriopathy in 1 patient with other vascular disease risk factors. All neurologic events resolved after stopping blinatumomab or ponatinib. The majority of patients were evaluated after one cycle (n=11, after cycle 2 n=3, after cycle 3 n=1). All but one patients (93%) obtained a cytologic complete remission (CR), of whom 12/14 (86%) achieved a complete molecular response. However, 2 patients were documented with CNS relapse at the time of blina/pona evaluation although in bone marrow molecular remission. Both obtained clearance of leukemic blasts after intrathecal infusion of chemotherapy. Then, 5 patients underwent allogeneic transplant (including 2 patients already allotransplanted before blina/pona) and 1 patient received donor lymphocyte infusion. Seven cases pursued maintenance therapy with ponatinib as single agent after stopping blinatumomab. With a median follow-up of 8 months (range: 2.6-30.2) for alive patients, median overall and leukemia-free survivals from first cycle of blina/pona were 8.5 months (range: 1.7-30.2) and 8 months (range: 1.3-30.2), respectively. At last follow-up (July 2018), only 4 relapses had occurred at a median of 3.3 months (range: 1.3-8.3) from first blina/pona cycle and 6 patients had died (3 bacterial infections, 1 fungal infection, 1 secondary cancer and 1 ALL relapse). All alive patients (n=9) but one (cytologic CR but detectable minimal residual disease) are in complete molecular response. Four patients are still under ponatinib medication at last follow-up. The combination of blinatumomab+ponatinib appears effective and tolerable in relapsed Ph1+ALL patients and may replace chemotherapy salvage regimens. The combination should be tested in first-line therapy in the future. Our results have also to be confirmed prospectively on a larger cohort of patients. Disclosures No relevant conflicts of interest to declare.

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SAR650984 (SAR) Directly Promotes Homotypic Adhesion-Related Multiple Myeloma (MM) Cell Death and SAR-Induced Anti-MM Activities Are Enhanced By Pomalidomide, More Potently Than Lenalidomide

Blood ◽

10.1182/blood.v124.21.2124.2124 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2124-2124 ◽

Cited By ~ 1

Author(s):

Hua Jiang ◽

Gang An ◽

Chirag Acharya ◽

Mike Y Zhong ◽

Ti Cai ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Caspase 3 ◽

Single Agent ◽

Annexin V ◽

Cell Killing ◽

Individual Agent ◽

Direct Cell ◽

Direct Killing

Abstract SAR650984 (SAR) is a naked humanized IgG1 monoclonal antibody (mAb) selectively targeting the membrane protein CD38 in early clinical development to treat multiple myeloma (MM) and other CD38+ hematological malignancies. SAR has demonstrated encouraging single agent activity in relapsed/refractory (R/R) MM patients (ASCO abstract #8532) and even better efficacy when combined with Dexamethasone and Lenalidomide (Len), without reaching a maximum tolerated dose in patients with heavily pretreated MM (ASCO Abstract #8512). It functions through multiple mechanisms including antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC), and direct killing against CD38-positive tumor cells including MM. Although SAR induces lysis of all CD38-expressing MM cell lines via ADCC, it only significantly induces direct killing of MOLP8 cells that express the highest CD38 surface density (~580,000/cell) among > 17 MM cell lines. We first sought to determine whether direct cell death induced by SAR depends on CD38 levels on MM cell membrane by generating RPMI8226 cells overexpressing CD38 (R-CD38) (Abstract #67338). R-CD38 cells express > 6-fold higher CD38 mRNA and surface protein levels than parental RPMI8226 cells (577,304/cell vs. 128,713/cell). Direct MM cell killing by SAR was determined using caspase 3/7 activity and CellTiter-Glo luminescent cell viability assays without goat anti-human IgG crosslinking, in the presence or absence of IL-6 or bone marrow stromal cells (BMSCs). Following overnight incubation, SAR significantly induced homotypic aggregation (HA) of R-CD38, but not control RPMI8226 cells, associated with dose-dependent activation of pro-apoptotic caspase 3/7 in R-CD38, but not control cells. Importantly, SAR decreased the viability of R-CD38, but not control cells, regardless of the presence of IL-6 or BMSCs. Direct cell death induced by SAR depends on SAR-induced HA in MM cells since SAR only blocked survival of R-CD38 and MOLP8 MM cells that show significant HA. Thus, direct apoptosis induced by SAR depends on the level of CD38 surface expression, which may contribute to clinical responses in R/R MM expressing higher CD38 levels. Next, we evaluated the combination effect of Len or Pomalidomide (Pom) with SAR on MM cells. BM mononuclear cells from MM patients were incubated with SAR (10 mg/ml) with or without 10 mM of Len or Pom overnight, followed by flow cytometric analysis to determine % Annexin V/PI staining of CD138+/BCMA+ MM cells. As expected, Pom alone induced slightly higher % of Annexin V+/PI+ MM cells than Len (41 + 1.8 % vs 49 + 1.5 %). Either combination further increased the % of double positive MM patient cells when compared with individual agent alone (from 40 + 2.1% to 70 + 3.1% combined with Len; from 40 + 2.1% to 86 + 3.4% combined with Pom). In addition, PBMC effectors from normal donors (n=4) were pretreated with Len or Pom (5 mM) for 3-7 days and used for SAR-mediated ADCC assays against MM cells (MM1S, MM1R, RPMI8226, R-38, MOLP8), with or without HS-5 or BMSCs from patients. Pom, more potently than Len, further increased SAR-induced MM cell lysis regardless of the presence of BMSCs. Moreover, additional pretreatment of MM cells with Pom overnight further enhanced SAR-induced ADCC by Pom-pretreated PBMC effectors. Both MOLP8 and R-CD38 are relative resistant to direct cytotoxicities induced by Len or Pom. Significantly, Pom, also more potently than Len, augmented direct toxicities induced by SAR in MOLP8 and R-CD38 MM cells. Taken together, we here demonstrate that SAR directly induces apoptosis of MM cells with higher CD38 levels; and that Pom, more effectively than Len, increases SAR-induced MM cell killing via apoptosis and ADCC. These data strongly support SAR as a monotherapy or in combination treatment to improve the outcome of MM patients. Disclosures Cai: Sanofi: Employment. Song:Sanofi: Employment. Yang:Sanofi: Employment. Adrian:Sanofi: Employment. Munshi:Celgene: Consultancy; Onyx: Consultancy; Janssen: Consultancy; Sanofi-Aventis: Consultancy; Ocopep: Consultancy, Equity Ownership, Patents & Royalties. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.

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Preclinical Evaluation of Tysabri as a Novel Adjuvant Therapy against Drug Resistant B-ALL.

Blood ◽

10.1182/blood.v114.22.3089.3089 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3089-3089 ◽

Cited By ~ 1

Author(s):

Yao-Te Hsieh ◽

Enzi Jiang ◽

Carlton Scharman ◽

Ella Waters ◽

Eugene Park ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Adjuvant Therapy ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Scid Mice ◽

Prolonged Survival ◽

Control Group ◽

Drug Resistant

Abstract Abstract 3089 Poster Board III-26 Novel treatment strategies for pediatric acute lymphoblastic leukemia (ALL) have turned a rapidly deadly diagnosis into a highly treatable entity, but we are still failing 25% of our pediatric ALL patients who die of recurrent ALL. Definitive studies have demonstrated that adhesion of leukemia and lymphoma cells to extracellular matrices or stromal cells protects them against the toxicity of cytoreductive chemotherapy drugs. In this context, a specific role for CD49d, a dominant adhesion molecule for normal lymphocytes, was demonstrated for acute myeloid leukemia (AML) and other malignant hematopoietic cells. The finding that CD49d blockade sensitizes AML cells to chemotoxicity may be of therapeutic potential, as is suggested by recent findings for AML cells engrafted in NOD/SCID mice. CD49d is and is similarly expressed on acute lymphoblastic leukemia (ALL) cells, but our knowledge about CD49d adhesion-mediated chemoprotection of B-ALL is limited. We hypothesized whether similar to primary AML blasts, xenografted ALL cells resistant to chemotherapy can be sensitized to chemotherapy by disrupting their CD49d-mediated adhesive interaction with stroma. To test our hypothesis we used as a CD49d inhibitor the humanized anti-human CD49d antibody natalizumab, or Tysabri®, which is in clinical use for the treatment of relapsing or refractory Multiple Sclerosis. To determine the potential of Tysabri as a single agent to decrease leukemia progression, we engrafted 5-7 weeks old NOD/SCID mice with primary drug resistant B-ALL labeled with lentiviral luciferase to allow monitoring of leukemia using noninvasive bioluminescent imaging. Tysabri administered upon detection of engraftment on Day15 post-injection of leukemia in the dose of either 1 mg (n=3) or 6 mg (n=3) led to remarkably slower leukemia progression regardless of the dose compared to the control group treated with saline only (n=2). Additional administration of Tysabri on day 29 and day 37 did not result in further containment of leukemogenesis but still showed a marked reduction in progression compared to the saline treated control group. In addition, we determined in vivo that a weekly administration of Tysabri in the dose of 5mg/kg/d resulted in prolonged survival compared to the treated control (p<0.05). Next, we assessed the effect of adjuvant anti-CD49d antibody-mediated dislodgement of ALL cells of drug resistant patients in combination with chemotherapy. The group treated for 4 weeks with chemotherapy including Vincristine, Dexamethasone and L-Asparaginase (VDL) in combination with Tysabri (5mg/kg/d) admistered once weekly showed decreased progression of leukemia and significantly prolonged survival (p<0.05) compared to the VDL only treated control group. No toxicity of Tysabri treatment was observed. Taken together, our data indicates the potential of Tysabri as a novel adjuvant therapy for treatment of drug resistant B-ALL. Given the availability of clinical-grade CD49d blocking antibody, clinical studies can follow immediately, should our hypothesis be confirmed in further in vitro an in vivo studies. Disclosures No relevant conflicts of interest to declare.

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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)

Blood ◽

10.1182/blood.v120.21.4865.4865 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4865-4865 ◽

Cited By ~ 1

Author(s):

Aradhana Awasthi Tiwari ◽

Janet Ayello ◽

Carmella van de Ven ◽

Danielle Glassman ◽

Anthony Sabulski ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Nk Cells ◽

Cell Lines ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Dismal Prognosis

Abstract Abstract 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) 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.

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Comparative Analysis on the Anti-Tumor Activity of Venetoclax and Obinutuzumab (VO) Versus Venetoclax and Rituximab (VR) in Primary CLL Cells, Ex Vivo

Blood ◽

10.1182/blood-2018-99-120188 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5558-5558

Author(s):

Madiha Iqbal ◽

Aarushi Sharma ◽

Alak Manna ◽

Sharoon Akhtar ◽

Taimur Sher ◽

...

Keyword(s):

Cell Death ◽

High Risk ◽

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

Single Agent ◽

Annexin V ◽

Intermediate Risk

Abstract INTRODUCTION: Treatment of chronic lymphocytic leukemia (CLL) has expanded significantly with the approval of multiple small molecule inhibitors. This is of great significance for patients with adverse cytogenetic features who tend to respond poorly to standard chemo-immunotherapy (CIT). While single agent ibrutinib and venetoclax (V) have shown high rates of overall response, complete remission and minimal residual disease (MRD) eradication rates remain low. This argues for further testing and development of various combination strategies. The MURANO trial compared venetoclax and rituximab (VR) versus bendamustine and rituximab (BR) in patients with relapsed/refractory CLL reporting clear superiority of VR over BR. MRD rates in the bone marrow were reported to be 27.3% for VR versus 1.5% for BR. Given much higher rates of MRD eradication with combination of small molecule inhibitors and monoclonal antibodies (mAb) compared to standard CIT, we performed a comparative investigation into the direct and immune-mediated cytolytic effects of VR versus V + Obinutuzumab (O, type II anti-CD20 mAb) in primary CLL cells and B-lymphoid cell lines. METHODS: CD19+ B-cells were isolated from PBMCs of CLL patients (N=3). For all experiments using primary CLL cells, concentration of VR and VO was 3nM (V) and 10ug /ml (R, O), respectively. For cell lines, VR and VO was used at 5uM (V) and 10ug /ml (R, O), respectively. Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by VR and VO was assessed in Calcein AM labeled CLL cells or cell lines co-cultured with healthy donor PBMCs (E:T ratio, 40:1); complement-dependent cytotoxicity (CDC) was measured using 10% serum from a healthy human donor. RESULTS: We assessed the ability of V+/-O or V+/-R to induce apoptotic cell death in the CD20+ BCWM.1 cell line (Waldenström's macroglobulinemia [WM] phenotype) and the MEC-1 cell line (B-PLL phenotype); with CD20- RPCI-WM1 (WM cells, negative control). Notably, Bcl-2 protein is expressed in all the aforementioned cell lines. We observed that single agent V, O and R induced ~30%, 61% and 13.64% annexin V/PI positivity in BCWM.1 cells, respectively. However, a significant degree of cell death was noted in VO-treated cells (~74%) compared to VR-treated cells (~40%) (p<0.01). Next, we examined for apoptosis in MEC1 cells and noted a similar trend; where the VO combination induced markedly more cell death (~71%) than VR (~57%). Contrastingly, in RPCI-WM1 cells neither single agent O or R could elicit >12% annexin V/PI positivity and where the addition of V increased apoptosis by only 3 - 4%. We also examined the apoptotic potential of VO or VR in tumor cells from low, intermediate and high-risk CLL patients. In low and intermediate-risk CLL cells from low and intermediate-risk patients, V alone induced ~30% cell death, which increased significantly with the addition of O (VO) to between 48 - 52%. Contrastingly, the combination of VR did not induce more than 29 - 32% apoptosis. In CLL cells from high-risk patient, we noted that exposure to single agent V induced ~ 28% cell death and in VO-treated cells, this number increased to 47%. We also examined for ADCC and CDC in the same cell lines and primary CLL cells. Despite considerable variability, single agent O and VO treatment of tumor cells resulted in greater ADCC than VR treatment. By contrast, in single agent R or VR-treated cells, more CDC was observed. CONCLUSION: Our preliminary investigation in VR- and VO-treated cell lines and primary CLL cells suggests the VO combination may be superior to VR in induction of direct tumor cell death. Mechanistic experiments underway will provide further insight and can aid in design of future VO-based clinical studies in CLL. Disclosures Ailawadhi: Pharmacyclics: Research Funding; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.

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Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Blood ◽

10.1182/blood.v122.21.1433.1433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1433-1433

Author(s):

Melanie Schirmer ◽

Manon Queudeville ◽

Luca Trentin ◽

Sarah M Eckhoff ◽

Lueder H Meyer ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Small Molecule ◽

Lymphoblastic Leukemia ◽

Xenograft Model ◽

Apoptosis Resistance ◽

Smac Mimetics ◽

Smac Mimetic

Abstract Intensified treatment of pediatric acute lymphoblastic leukemia (ALL) has lead to increased survival rates of about 80%, however therapy fails in the remaining patients leading to relapse of the disease associated with inferior prognosis. Because treatment failure is, at least in part, due to defects in apoptosis programs, novel therapeutic strategies that counter apoptosis resistance are needed. “Inhibitor of Apoptosis” (IAP) proteins block the apoptosis machinery at a central point and are highly expressed in acute leukemias, thereby providing a target structure for therapeutic intervention. Molecules antagonizing these apoptosis inhibitors, so called SMAC-mimetics, therefore provide a promising strategy to overcome apoptosis deficiency and effectively treat high risk ALL. In this study, we investigated the effects of the small molecule SMAC-mimetic BV6 (kindly provided by Genentech) in B cell precursor- (BCP-) ALL. BV6 showed a clear induction of cell death at nanomolar concentrations in ALL cell lines. ALL cells sensitive for SMAC-mimetic induced cell death showed rapid cIAP degradation, NFkB activation and secretion of TNF-alpha (TNF-a). Interestingly, mitochondrial perturbation and caspase activation could be inhibited by the soluble TNF-a receptor Etanercept indicating the induction of a TNF-a dependent feed forward loop by the SMAC-mimetic BV6. In addition to cell lines, we investigated the effects of BV6 on a series of 42 primary ALL samples isolated from ALL bearing mice of established patient derived NOD/SCID/huALL xenograft leukemias. Intriguingly, upon treatment with the small molecule SMAC mimetic BV6, induction of cell death was observed in a majority of 70% of all individual patient-derived leukemias and BV6 induced cell death was inhibited by Etanercept demonstrating TNF-a dependency also in primary ALL. We previously described that rapid engraftment of ALL cells transplanted onto NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in the ALL cells and indicative for early patient relapse. Importantly, primary xenograft ALL samples with a TTLshort/early relapse phenotype showed increased cell death upon treatment with SMAC-mimetic BV6 and activation of the constitutive deficient apoptosis signaling pathway, demonstrating that SMAC-mimetics induce intact apoptosis signaling in former apoptosis resistant primary ALL cells. Based on theses findings, we further evaluated the in vivo effectivity of the SMAC-mimetic BV6 on high risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. ALL bearing recipients were treated with either BV6 or solvent for a given time of two weeks and further investigated for the presence of leukemia. Most interestingly, a significant delay of post-treatment leukemia reoccurrence was observed upon BV6 in vivo treatment along with a profound reduction of tumor load in the recipients compared to solvent treated animals. In a clinical setting, high-risk disease is unlikely to be treated by one compound alone. Therefore, we combined BV6 with multidrug chemotherapy resembling ALL induction treatment and observed a significant delay of ALL reoccurrence and prolonged survival of animals treated with the combination of the SMAC-mimetic and chemotherapy in contrast to chemotherapy alone. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death sensitizing effect of BV6, both in single treatment and in combination with chemotherapy. This indicates that BV6 induced apoptosis sensitization involves signaling via TNF-a and thereby provides a potential biomarker for the identification of patients who would benefit from SMAC-mimetic treatment. Taken together, we show that the small molecule SMAC-mimetic BV6 induces cell death via a TNF-a loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL. Thus, induction of cell death by new generation small molecule SMAC-mimetics provides a promising novel strategy for targeted therapy of high-risk acute lymphoblastic leukemia and involvement of TNF-a signaling in BV6-sensitive patients points to its potential use as biomarker indicating effective cell death sensitization. Disclosures: No relevant conflicts of interest to declare.

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A Novel DLL1-Anti-CD19 Chimeric Protein Effectively Targets Notch Activation to B-Cell Acute Lymphoblastic Leukemia and Induces Cell Death

Blood ◽

10.1182/blood.v126.23.4833.4833 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4833-4833

Author(s):

Rebecca E Wiersma ◽

Sankaranarayanan Kannan ◽

Srinivas S. Somanchi ◽

Lizhi Zeng ◽

Patrick A. Zweidler-McKay

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Notch Signaling ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Chimeric Protein ◽

Notch Ligand ◽

Cell Counts ◽

Notch Receptors

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most prevalent form of cancer in children, and those who relapse continue to have poor survival. Therefore, the development of improved and specifically targeted treatment options is vital. The Notch pathway has been shown to act as a tumor suppressor in B-ALL via cell type specific induction of growth-inhibiting and pro-apoptotic pathways. In this study, we aim to therapeutically activate Notch signaling in B-ALL via targeting the Notch ligand DLL1 to B cells using an anti-CD19 scFv chimeric protein. Methods: A soluble chimeric protein composed of the extracellular domain of the Notch ligand DLL1 linked to a validated anti-CD19 scFv was isolated from HEK-293 producer cells. Human B-ALL (SB, Nalm6) and T-ALL (Jurkat) lines were treated with DLL1-anti-CD19scFv chimeric protein, and expression of the Notch target gene HES1 and effects on cell growth and survival were measured. Results: Both B- and T-ALL lines express Notch1 and Notch2 receptors on the cell surface. Exposure of B-ALL cells to DLL1-anti-CD19scFv chimeric protein led to an increase of Notch signaling, measured via 3-14 fold increase in HES1 mRNA expression. As expected, surface expression of the Notch receptors decreased upon chimera exposure, as Notch receptors are cleaved and destroyed upon activation. Importantly, exposure of the B-ALL lines to the chimeric protein led to a maximum 60% decrease in cell counts over 3-4 days, in contrast to T-ALL, where exposure did not effect growth significantly. Conclusions: This study demonstrates that Notch signaling can be feasibly activated in human B-ALL cells through a soluble DLL1-anti-CD19 scFv chimeric protein. Activation of Notch signaling via this method leads to growth inhibition and cell death in B-ALL, but not T-ALL cells. Based on our findings, we suggest that this soluble DLL1-anti-CD19 chimera may be a potential therapeutic approach for B-ALL, and further in vivo testing is warranted. Disclosures No relevant conflicts of interest to declare.

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Changes in cell death of peripheral blood lymphocytes isolated from children with acute lymphoblastic leukemia upon stimulation with 7 Hz, 30 mT pulsed electromagnetic field

Cellular & Molecular Biology Letters ◽

10.1515/cmble-2015-0006 ◽

2015 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Jolanta Kaszuba-Zwoińska ◽

Magdalena Ćwiklińska ◽

Walentyna Balwierz ◽

Paulina Chorobik ◽

Bernadeta Nowak ◽

...

Keyword(s):

Cell Death ◽

Electromagnetic Field ◽

Acute Lymphoblastic Leukemia ◽

Propidium Iodide ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Pulsed Electromagnetic Field ◽

Pulsed Electromagnetic ◽

In Cells

AbstractPulsed electromagnetic field (PEMF) influenced the viability of proliferating in vitro peripheral blood mononuclear cells (PBMCs) isolated from Crohn’s disease patients as well as acute myeloblastic leukemia (AML) patients by induction of cell death, but did not cause any vital changes in cells from healthy donors. Experiments with lymphoid U937 and monocytic MonoMac6 cell lines have shown a protective effect of PEMF on the death process in cells treated with death inducers.The aim of the current study was to investigate the influence of PEMF on native proliferating leukocytes originating from newly diagnosed acute lymphoblastic leukemia (ALL) patients.The effects of exposure to PEMF were studied in PBMCs from 20 children with ALL. PBMCs were stimulated with three doses of PEMF (7 Hz, 30 mT) for 4 h each with 24 h intervals. After the last stimulation, the cells were double stained with annexin V and propidium iodide dye to estimate viability by flow cytometric analysis.The results indicated an increase of annexin V positive as well as double stained annexin V and propidium iodide positive cells after exposure to threefold PEMF stimulation.A low-frequency pulsed electromagnetic field induces cell death in native proliferating cells isolated from ALL patients. The increased vulnerability of proliferating PBMCs to PEMF-induced interactions may be potentially applied in the therapy of ALL.The analysis of expression of apoptosis-related genes revealed changes in mRNA of some genes engaged in the intrinsic apoptotic pathway belonging to the Bcl-2 family and the pathway with apoptosis-inducing factor (AIF) abundance upon PEMF stimulation of PBMCs.

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Arsenic Trioxide Sensitizes Glucocorticoid-Resistant T-Cell Acute Lymphoblastic Leukemia to Dexamethasone through Inactivation of AKT and Downregulation of XIAP.

Blood ◽

10.1182/blood.v108.11.2598.2598 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2598-2598

Author(s):

Beat C. Bornhauser ◽

Laura Bonapace ◽

Felix K. Niggli ◽

Beat W. Schaefer ◽

Jean-Pierre Bourquin

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Arsenic Trioxide ◽

Low Dose ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Chemotherapeutic Agents ◽

Parp Cleavage ◽

Akt Phosphorylation

Abstract Arsenic trioxide (ATO) is a very effective agent for the treatment of acute promyelocytic leukemia (APL). Because of its pro-apoptotic activity already at low dosage, ATO may be beneficial for combination treatment of drug-resistant disease to augment the effect of other chemotherapeutic agents. In childhood acute lymphoblastic leukemia (ALL), de novo resistance to glucocorticoids (GC) is markedly associated with poor outcome. As a mechanism of action, GCs induce apoptosis in steroid-sensitive leukemic cells. Recent evidence suggests that GC-resistance is associated with altered levels of pro- and anti-apoptotic regulatory proteins. We therefore evaluated the potential of ATO as a chemo-sensitizer in a model of GC-resistant ALL. As expected, ATO effectively induced cell death in ALL cell lines and in primary ALL patient samples at clinically relevant concentrations (IC50 values between 0.4–1.2 uM). Interestingly, ATO re-sensitized the GC-resistant ALL lines CEM C1-15, MOLT-4 and Jurkat to dexamethasone at doses that do not affect cell survival as single agent. The same effect was observed using primary cells from two patients with GC-resistant T-cell ALL. The sensitizing effect of ATO could not be observed in combination with the standard ALL chemotherapeutic agents daunorubicin, asparaginase and vincristine, suggesting that a mechanism specifically relevant for GC-resistance is involved. ATO, alone or at lower dose in combination with dexamethasone, induced cell death by activating caspase-3 and −8, which resulted in PARP cleavage. We then investigated the MAPK and PI3K/AKT pathways as candidate mechanisms of action with key apoptosis regulators such as Bad and Bcl-2 as targets. The MEK1/2 inhibitor PD98059 only marginally augmented the response to dexamethasone, suggesting that this pathway would not play a major role for GC sensitization. In contrast, we found that low dose ATO treatment resulted in a rapid decrease of AKT phosphorylation at the activating serine 473 phosphorylation site, while AKT levels remained unaffected. Concomitantly, the AKT targets Bad and XIAP appeared to be modulated. The pro-apoptotic regulator Bad was induced, and its phosphorylation was decreased. Downregulation of Bad mediated by siRNA partly rescued cells from ATO-induced cell death indicating that Bad might have a sensitizing role in ATO-mediated apoptosis. Moreover, ATO induced downregulation of the NF-kB target XIAP. The protein levels of other NF-kB targets such as Bcl-2 and Bcl-xL were unchanged. Downregulation of XIAP could be due to decreased protein stability resulting from reduced XIAP phosphorylation by AKT. The effects of low dose ATO on AKT phosphorylation as well as Bad and XIAP levels were more pronounced when cells were treated in combination with dexamethasone. Ongoing experiments will further define the role of XIAP and NF-kB as specific therapeutic targets in this model. These results indicate that resistance to GCs is - at least in part - mediated through apoptosis regulators in ALL. Our data provide evidence that ATO may be beneficial as sensitizing agent in defined subgroups of ALL patients, next to its role in APL and multiple myeloma treatment.

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