Unique and Potent Tumor Specific Antibodies in Graft Versus Leukemia Responses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3826-3826
Author(s):  
Marijn A. Gillissen ◽  
Etsuko Yasuda ◽  
Sophie Levie ◽  
Arjen Q Bakker ◽  
Martijn Kedde ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
High Risk ◽  
B Cell ◽  
Cell Lines ◽  
Surface Antigens ◽  
Antibody Responses ◽  
Hematopoietic Stem ◽  
Specific Antibodies ◽  
Graft Versus Leukemia

Abstract Introduction Acute myeloid leukemia (AML) is a high-risk malignancy with a poor prognosis. Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative if it induces a potent graft versus leukemia (GvL) response. GvL responses and graft versus host disease (GvHD) are typically considered T cell mediated, because T cell depletion from hematopoietic stem cell grafts reduces the risk of GvHD at the cost of leukemia relapse. In addition, depletion of B-lymphocytes with rituximab has led to amelioration of GvHD in a number of studies suggesting that B cells are also important in the pathophysiology of GvHD and, in analogy, in GvL responses. However, the characteristics of the antibodies produced by these leukemic specific B cells have not yet been studied. Methods We selected three patients with high-risk myelomonocytic leukemia who remained disease free years after allogeneic HSCT, from whom we established clonal human B cell lines, using a unique and innovative technology that was developed in our laboratory (Kwakkenbos ea, Nat Med 2010). These B cell lines, that concomitantly express immunoglobulin on their membranes and secrete antibodies, were used to select antibodies specific for cell surface antigens on AML cell lines with similar morphologic and immunophenotypic characteristics as the patients’ leukemic blasts, using a FACS based assay. Results From each patient, several AML specific B cell clones were retrieved. Their antibodies recognized surface antigens on primary AML blasts derived from multiple patients and on AML cell lines, but not on healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. The majority of the antibodies were of the IgG3 isotype. Approximately 40% of the AML-specific antibodies induced direct death of cultured AML cell lines and of primary AML blasts. The cell death pathway induced by these cytotoxic antibodies was oncotic rather than apoptotic. Conclusion Our data demonstrate that high-risk AML patients with a potent GvL response mount robust antibody responses against surface antigens that are specifically expressed on tumor cells and binding of these antibodies induces direct cell death. The targets recognized by the recovered antibodies are also expressed on leukemic blast from other patients suggesting evidence for a common immune mechanism responsible for AML clearance. Our findings suggest that antibody responses are important in GvL and open up new ventures for specific antibody treatment of AML patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals GSTM1 and GSTT1 double null genotypes determining cell fate and proliferation as potential risk factors of relapse in children with hematological malignancies after hematopoietic stem cell transplantation

2021 ◽  
Author(s):  
Simona Jurkovic Mlakar ◽  
Chakradhara Rao Uppugunduri Satyanarayana ◽  
Tiago Nava ◽  
Vid Mlakar ◽  
Hadrien Golay ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Purpose This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models. Methods GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR–Cas9 gene-edited THP1 leukemia cell lines. Results Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76–15.42; p = 1.9 × 10–5]). BU-induced cell death preferentially in THP1GSTM1(non−null) and LCLsGSTM1(non−null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation.

scholarly journals GSTM1 and GSTT1 Double Null Genotypes Determining Cell Fate and Proliferation as Potential Risk Factors of Relapse in Children with Hematological Malignancies after Stem Cell Transplantation.

2021 ◽  
Author(s):  
Simona Jurkovic Mlakar ◽  
Chakradhara Rao Uppugunduri Satyanarayana ◽  
Tiago Nava ◽  
Vid Mlakar ◽  
Hadrien Golay ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Leukemia Cell ◽  
Hematopoietic Stem ◽  
Glutathione S Transferases ◽  
The Impact

Abstract Background: Relapse is the major cause of treatment failure in children with hematological malignancies (HMs) undergoing busulfan (BU)- based allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferases (GSTs) isoforms that participate in BU detoxification and protect cells against stress and cell death may be linked to post-HSCT outcomes. This study aimed to retrospectively evaluate the genetic association of null variants of Glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with HMs undergoing BU- containing allogeneic HSCT and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized and tumor lymphoblastoid cell lines (LCLs).Methods: GSTM1- and GSTT1- null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1- null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell-death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. Results: Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76 - 15.42; p= 1.9 x 10-5]). BU induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. Conclusion: The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation. Trial registration: ClinicalTrials.gov identifier: NCT01257854, Registered February 2008 – retrospectively registered.

scholarly journals Synergistic Activity of the MCL-1-Specific Inhibitor S63845 with Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1416-1416
Author(s):  
Felix Seyfried ◽  
Felix Stirnweiß ◽  
Stefan Köhrer ◽  
Klaus-Michael Debatin ◽  
Lüder Hinrich Meyer
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Specific Inhibitor ◽  
Synergistic Activity ◽  
Cell Precursor

Abstract Deregulated cell death and survival pathways contribute to leukemogenesis and treatment failure of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patients. The intrinsic apoptosis pathway is regulated at the mitochondrial level by different pro- and anti-apoptotic molecules. Members of the BCL-2 family are key regulators of mitochondrial apoptosis signaling. Pro-apoptotic BH3-only proteins like BIM and BID activate pro-death proteins such as BAX and BAK leading to cell death. Anti-apoptotic BCL-2 family members including BCL-2, BCL-XL and MCL-1 bind to and sequester pro-apoptotic molecules, prevent activation of pro-death proteins and counter-regulate apoptosis induction. Small molecule inhibitors have been developed that block binding to anti-apoptotic molecules like BCL-2, leading to release of pro-apoptotic proteins and cell death induction. In particular, the BCL-2-specific inhibitor venetoclax (VEN) has demonstrated substantial anti-cancer activity and became an approved drug for the treatment of CLL patients. Investigating different, individual BCP-ALL samples, we and others recently identified heterogeneous sensitivities for VEN, suggesting that BCP-ALL cells might also depend on other pro-survival BCL-2 family proteins including MCL-1, leading to VEN insensitivity and resistance. A novel BH3-mimetic, S63845, that selectively targets MCL-1 has been reported. Here, we assessed the activity of S63845 and addressed a potential synergism of simultaneous blockage of BCL-2 and MCL-1 by VEN and S63845 (S) in BCP-ALL. The activity of the MCL-1 inhibitor was analyzed in a panel of BCP-ALL cell lines (N=6) and a series of primary, patient-derived BCP-ALL primograft samples (N=27) determining half-maximal effective concentrations (EC50) upon exposure to increasing concentrations of S and analysis of cell death induction. We observed heterogeneous sensitivities to S with EC50 values ranging from 16 nM to almost 10 µM. Protein expression of MCL-1 and other BCL-2 family members BCL-2, BCL-XL and BCL-W was assessed by western blot analysis and quantified, however neither association of MCL-1 levels nor expression of the other regulators and S sensitivity was found in cell lines and primograft leukemias. Moreover, we also compared sensitivities for both inhibitors but found independent activities of S and VEN in individual ALL samples. Next, we addressed the role of MCL-1 for VEN sensitivity and generated two MCL-1 knock out BCP-ALL cell lines by CRISPR/Cas9 gene editing. In both lines, clearly increased VEN sensitivities were observed upon depletion of MCL-1, indicating that MCL-1 is contributing to activity of the BCL-2 inhibitor VEN. Based on these findings, we investigated the effects of pharmacological MCL-1 inhibition for VEN sensitivity and incubated all 6 cell lines with VEN and S at increasing concentrations and observed clear synergistic effects upon combined BCL-2 and MCL-1 inhibition indicated by combination indices (CI) below 0.1. Moreover, we investigated 7 primograft BCP-ALL samples and found that MCL-1 inhibition by S clearly synergized with VEN activity (CI < 0.3). To investigate the anti-leukemia activity of co-targeting BCL-2 and MCL-1 in vivo in a pre-clinical setting, a high-risk leukemia derived from an infant, MLL/ENL rearranged pro-B ALL case was transplanted onto NOD/SCID mice. Upon ALL manifestation (presence of >5% human blasts in blood), recipients were treated with either VEN, S, the combination of both, or vehicle for 10 days. After treatment, leukemia loads were analyzed showing significantly reduced loads in the co-treated group as compared to vehicle, VEN or S alone in spleen, bone marrow, and central nervous system (p-values < 0.05), indicating synergistic activity of co-inhibition of BCL-2 and MCL-1 in vivo. Taken together, our data show heterogeneous sensitivity of individual BCP-ALL samples to MCL-1 inhibition by S, which is not associated with MCL-1 protein expression levels or VEN sensitivity. Both, genetic depletion and inhibition of MCL-1 by S synergizes with VEN leading to increased anti-leukemia activity in vitro and ex vivo. Importantly, co-targeting BCL-2 and MCL-1 significantly reduced leukemia infiltration in spleen, BM and CNS in a pre-clinical model of high-risk BCP-ALL, warranting further evaluation and possible clinical application of targeting MCL-1 alone and in combination with BCL-2 inhibition. Disclosures No relevant conflicts of interest to declare.

Donor-Derived B Cells Produce Potent AML-Specific Antibodies That Recognize Novel Tumor-Specific Antigens and Mediate Graft-Versus-Leukemia Immunity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3146-3146 ◽  
Author(s):  
Marijn Aletta Gillissen ◽  
Martijn Kedde ◽  
Etsuko Yasuda ◽  
Sophie E. Levie ◽  
Arjen Q. Bakker ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
B Cells ◽  
Stem Cell Transplantation ◽  
Tumor Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Allogeneic Hsct ◽  
Graft Versus Leukemia ◽  
Specific Antigens

Abstract Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regression. While the role of T cells in such graft versus leukemia (GvL) immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic hematopoietic stem cell transplantation (HSCT) has been demonstrated. The function of these antibodies remains to be established though. Here we tested the hypothesis that B cells contribute to GvL. We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from the peripheral blood of these patients with Bcl-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Target identification analyses revealed two novel, AML-specific surface proteins as primary targets of these antibodies. These newly identified AML-specific antigens are widely expressed on different types of AML, including blasts of the last 17 AML patients that entered our clinic of whom diagnosis material was made available for this project. Strikingly, antibody binding to one of these targets induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37o C and 4o C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Other antibodies induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC). Together these data indicate that tumor selective antibodies are elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct and indirect cytotoxic activities against tumor cells of these antibodies suggest that they contribute to the GvL response. Disclosures No relevant conflicts of interest to declare.

GSTM1 and GSTT1 Double Null Genotypes Determining Cell Fate and Proliferation as Potential Risk Factors of Relapse in Children With Hematological Malignancies After Stem Cell Transplantation. On Behalf of the Pediatric Disease Working Party of the European Society for Blood and Marrow Transplantation

2021 ◽  
Author(s):  
Simona Jurkovic Mlakar ◽  
Chakradhara Rao Uppugunduri Satyanarayana ◽  
Tiago Nava ◽  
Vid Mlakar ◽  
Hadrien Golay ◽  
...  
Keyword(s):  
Cell Death ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Hematological Malignancies ◽  
Leukemia Cell ◽  
Hematopoietic Stem ◽  
Glutathione S Transferases ◽  
The Impact

Abstract BackgroundRelapse is the major cause of treatment failure in children with hematological malignancies (HMs) undergoing busulfan (BU)- based allogeneic hematopoietic stem cell transplantation (HSCT). Glutathione S-transferases (GSTs) isoforms that participate in BU detoxification and protect cells against stress and cell death may be linked to post-HSCT outcomes. This study aimed to retrospectively evaluate the genetic association of null variants of Glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with HMs undergoing BU- containing allogeneic HSCT and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST-gene edited cell models.MethodsGSTM1- and GSTT1- null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1- null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell-death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. ResultsCarrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76 - 15.42; p= 1.9 x 10-5]). BU induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. ConclusionThe clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation. Trial registrationClinicalTrials.gov identifier: NCT01257854, Registered February 2008 – retrospectively registered.

scholarly journals FOXO1 Is Involved in the Regulation of B-Cell Precursor Acute Lymphoblastic Leukemia Survival and Serves As a Novel Target for Directed Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4020-4020
Author(s):  
Salih Demir ◽  
Fan Wang ◽  
Franziska Gehringer ◽  
Clarissa Weitzer ◽  
Klaus-Michael Debatin ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Precursor ◽  
Free Survival

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric and adolescent malignancy. Although current treatment provides five-year event-free survival, in up to 20% conventional chemotherapy fails resulting in relapse with inferior prognosis. FOXO1 is a member of the forkhead family of transcription factors, which is preferably expressed in B-cells with high expression at the early B-cell stage. FOXOs are involved in several cellular processes including cell death and proliferation, anti-cancer drug resistance and protection from oxidative stress. Since FOXO1 can enhance tumor growth and potentiate metastasis, we aimed to investigate the effects of FOXO1 inactivation on B-cell precursor (BCP)-ALL, including preclinical in vivo evaluation. FOXO1 expression levels were compared among 497 cancer samples using the Genevestigator online software. Expression of FOXO1 in BCP-ALL was significantly higher than in any of the other cancer types. Next, we investigated FOXO1 expression and subcellular localization in 3 BCP-ALL cell lines by cellular fractionation and fluorescent microscopy. Both methods showed localization of FOXO1 in the nucleus, indicating transcriptionally active FOXO1 in BCP-ALL. In order to study the potential anti-tumor effect of FOXO1 repression, we investigated genetically modified, FOXO1 deficient BCP-ALL cell lines (n=5) and observed no cell death induction in control transduced cells, in contrast to a clear reduction of cell viability of up to 80% upon FOXO1 knock-down, clearly indicating dependency of BCP-ALL cells on FOXO1. Moreover, lentiviral mediated FOXO1 knockdown did not induce cell death in the Hodgkin's lymphoma cell line cHL, suggesting a BCP-ALL specific importance for FOXO1. Based on these results indicating the importance of FOXO1 expression for BCP-ALL maintenance, we investigated the feasibility of pharmacological interference with FOXO1. Exposure of 7 BCP-ALL, 4 T-ALL, 3 B-cell NHL, 2 DLBCL and 3 cHL cell lines to the small molecular weight FOXO1 inhibitor AS1842856 showed effectivity in BCP-ALL lines, reflected by significantly higher half maximal inhibitory concentrations (IC50) by MTT test. The most sensitive cell line was the BCP-ALL line RS4;11, while the cHL cell line SUP-HD1 showed insensitivity for FOXO1 inhibition (IC50: 3 nM and 26 µM), again indicating that BCP-ALL is particularly dependent on FOXO1 activity. Caspase 3 cleavage detected upon exposure to AS1842856 showed induction of apoptosis as mechanism of cell death. Furthermore, we evaluated the sensitivity of primary BCP-ALL primograft samples (n=9) exposing the ALL cells to increasing pharmacologically relevant concentrations of AS1842856. The inhibitor increased cell death as measured by flow cytometry (FSC/SSC criteria) in all of the samples tested in a time and dose dependent manner. Importantly, FOXO1 inhibition also showed activity on high risk leukemias including MLL-rearranged and early or second-relapse cases. Moreover, we investigated the in vivo effectivity of AS1842856. Two different patient derived leukemias were transplanted onto NOD/SCID mice and upon leukemia manifestation vehicle or AS1842856 was administered for a time of 11 days. At the end of the experiment, all mice were sacrificed and tumor loads were quantified in spleen, bone marrow and central nervous system (CNS). Importantly, tumor loads of all compartments and spleen sizes were significantly reduced in AS1842856 treated animals (p=0.028, U-test). Moreover, in an early-relapse sample leukemia-free survival upon AS1842856 treatment was evaluated. Mice were treated by vehicle or AS1842856 (n=10/group) during 11 days. Leukemia-free survival was significantly prolonged in mice which received AS1842856 (p=0.003, Log-rank test). Taken together, we show that the active form of FOXO1 is highly expressed in BCP-ALL cells as compared to other cancers, and that viability of BCP-ALL cells is regulated by FOXO1 activity. Importantly, silencing or pharmacological inhibition of FOXO1 induces cell death in BCP-ALL primogafts including high risk cases, both ex vivo and preclinically in vivo. Thus, targeting FOXO1 provides a promising novel strategy for therapeutic intervention in these high-risk subtypes of BCP-ALL. Disclosures No relevant conflicts of interest to declare.

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