Generation of lymphocytes potentiated against leukemic lymphoblasts by stimulation using leukemic cell lysate-pulsed dendritic cells in patients with acute lymphoblastic leukemia and measurement of in vitro anti-leukemic cytotoxicity

Acute Lymphoblastic Leukemia (ALL) is the most common neoplasm in children, but the mechanisms underlying leukemogenesis are poorly understood, despite the existence of several theories regarding the mechanics of leukemic cell proliferation. However, with the advent of new biological principles, it appears that a systems approach could be used in an effective search of global patterns in biological systems, so as to be able to model the phenomenon of proliferation and gain a better understanding of how cells may progress from a healthy to a diseased state. This chapter reviews the current knowledge on proliferation dynamics, along with a discussion of the several existing theories on leukemogenesis and their comparison with the theories governing general oncogenesis. Furthermore, the authors present some “in-house” experimental data that support the view that it is possible to model leukemic cell proliferation and explain how this has been performed in in vitro experiments.

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Adoptive Transfers of Plasmacytoid Dendritic Cells after Hematopoietic Stem Cell Transplantation Decrease the Risk Acute Lymphoblastic Leukemia Relapse without Increasing the Risk of Graft-Versus-Host-Disease

Blood ◽

10.1182/blood-2018-99-111130 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2053-2053 ◽

Cited By ~ 1

Author(s):

Sabine Herblot ◽

Valérie Paquin ◽

Paulo Cordeiro ◽

Michel Duval

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Stem Cell ◽

Acute Lymphoblastic Leukemia ◽

Nk Cell ◽

Lymphoblastic Leukemia ◽

Hematopoietic Stem ◽

Graft Versus Host ◽

Antigen Presenting

Abstract Despite advances in chemotherapy and hematopoietic stem cell transplantation (HSCT), the outcome of children with relapsed acute lymphoblastic leukemia (ALL) has not significantly improved over the last 2 decades. About 50% of children with relapsed leukemia still die from their disease and ALL is still the first cause of death by cancer in children. A new hope of cure for patients with chemo-resistant cancers has emerged with the development of cancer immunotherapy. However, the major risk of post-transplant immunotherapy is the exacerbation of life-threatening Graft-versus-Host Disease (GvHD) mediated by donor-derived T cells. We therefore explored the avenue of innate immune stimulation. Several reports have demonstrated that activated Natural Killer (NK) cells can control acute myeloid leukemia (AML) in transplanted patients, whereas ALL is deemed to be resistant to NK cell killing. We recently challenged this paradigm and demonstrated that the stimulation of NK cells with third-party activated plasmacytoid dendritic cells (pDC) killed most ALL cell lines and patient-derived ALL blasts. We further demonstrated the efficacy of pDC adoptive transfers to cure ALL in a humanized mouse model of HSCT. Collectively, these results uncovered for the first time the unique therapeutic potential of activated pDC as immunotherapeutic tools to stimulate NK cell anti-leukemic activity early after HSCT. The next step toward the clinical translation of pDC-based post-transplant immunotherapy is to verify that adoptive transfers of pDC do not stimulate T cells nor exacerbate GvHD in the presence of mature T cells. We designed a GMP-compliant method for in vitro expansion and differentiation of cord blood progenitors giving rise to sufficient numbers of pDC for adoptive transfers in patients. We showed that after Toll-like receptor (TLR) stimulation, these in vitro differentiated pDC displayed a phenotype of interferon producing cells (CD80neg PDL-1+) but not of antigen presenting cells (CD80+PDL-1neg). Accordingly, in vitro mixed lymphocyte reactions with purified allogeneic T cells demonstrated that TLR-activated pDC induced very low allogeneic T cell proliferation as compared with bona fide antigen presenting cells such as myeloid dendritic cells (mDC - CD11c+) or monocyte-derived dendritic cells (mo-DC) (Figure 1A). To test whether activated pDC could exacerbate GvHD in the presence of mature T cells, we used a xenoGvHD model in which human peripheral blood mononuclear cells (PBMC) were injected in immune-deficient mice (Nod/Scid/gRc-/-, NSG). We monitored GvHD 3-times a week according to a GvHD-assessment scale as previously described. Overt GvHD was characterized by cutaneous and intestinal lesions, weight loss and high numbers of human CD3+ cells in peripheral blood. Mice were sacrificed when endpoints were reached and GvHD was confirmed by immunohistochemistry and flow cytometry. Five weekly injections of TLR-activated in vitro differentiated pDC did not accelerate the GvHD onset and the severity of the lesions were not increased. We did not either observe any difference in survival between control and pDC-treated groups (Figure 1B). Collectively, our results indicate that TLR-activated pDC do not stimulate allogeneic T cells and do not increase the risk of acute GvHD in a mouse model of xenoGvHD. We therefore expect this novel pDC-based immunotherapy to be safe for transplanted patients. These data open the way for the next step: a Phase I clinical trial of in vitro differentiated pDC after transplantation for leukemia. Disclosures No relevant conflicts of interest to declare.

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TPEN Selectively Eliminates Lymphoblastic B cells from Bone Marrow Pediatric Acute Lymphoblastic Leukemia Patients

10.20944/preprints202103.0611.v2 ◽

2021 ◽

Author(s):

Miguel Mendivil-Perez ◽

Carlos Velez-Pardo ◽

Lina Maria Quiroz-Duque ◽

Alexandra Restrepo-Rincon ◽

Natalia Andrea Valencia-Zuluaga ◽

...

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Anticancer Agents ◽

Pediatric Patients ◽

De Novo ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemia Cells

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages of differentiation. Despite advances in treatment, a significant percentage of pediatric patients with precursor B-ALL still relapse. Therefore, alternative therapies are needed to improve the cure rates for pediatric patients. TPEN (N, N, N’, N’-tetrakis(2-pyridylmethyl)-ethylenediamine).is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, it has been suggested that TPEN could be a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like model, for example, the bloodstream and bone marrow (BM), in vitro. This investigation is an extension of a previous study that dealt with the effect of TPEN on ex vivo isolated/purified refractory B-ALL cells. Here, we evaluated the effect of TPEN on whole BM from nonleukemic patients (control) or pediatric patients diagnosed with de novo B-ALL or refractory B-ALL cells by analyzing the hematopoietic cell lineage marker CD34/CD19. Although TPEN was innocuous to nonleukemic BM (n=3), we found that TPEN significantly induced apoptosis in de novo (n = 5) and refractory B-ALL (n = 6) leukemic cell populations. Moreover, TPEN significantly increased the counts of cells positive for the oxidation of the stress sensor protein DJ-1, a sign of the formation of H2O2, and significantly increased the counts of cells positive for the pro-apoptotic proteins TP53, PUMA, and CASPASE-3 (CASP-3), indicative of apoptosis, in B-ALL cells. We demonstrate that TPEN selectively eliminates B-ALL cells independent of age, diagnosis status (de novo or refractory), sex, karyotype, or immunophenotype. Understanding TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic oxidation-inducing anticancer agents.

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Prednisolone Resistance in Pediatric Acute Lymphoblastic Leukemia Can Be Synergistically Overcome by Inhibition of Anti-Apoptotic MCL1 and Glycolysis

Blood ◽

10.1182/blood.v120.21.3528.3528 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3528-3528

Author(s):

Ingrid M. Ariës ◽

Bo R. Hansen ◽

Troels Koch ◽

William E. Evans ◽

Rob Pieters ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Glucose Consumption ◽

Leukemic Cells ◽

Anaerobic Glycolysis ◽

Bcl2 Family ◽

Protein Levels ◽

Pediatric All

Abstract Abstract 3528 Background: Unsuccessful treatment of pediatric precursor B acute lymphoblastic leukemia (ALL) can be ascribed to cellular resistance to antileukemic drugs. In particular, resistance towards prednisolone is associated with poor prognosis in pediatric ALL. For three reasons, we hypothesized that anti-apoptosis sustained by the BCL2 family member MCL1 and glycolysis are linked processes and concomitantly induce resistance to prednisolone: 1) Glycolysis and apoptosis are closely related survival pathways both associated with prednisolone resistance, 2) Increased glucose metabolism has been directly linked to MCL1 stabilization and attenuation of apoptosis, and 3) BCL2 family members can adjust oxidative phosphorylation, a process that together with anaerobic glycolysis, is responsible for cellular respiration and ATP production. In this study, we functionally determined the synergistic contribution of MCL1 and glycolysis to prednisolone resistance in childhood ALL. Methods: Leukemic cells of pediatric ALL patients, >90% blasts, were treated in vitro with prednisolone for 48 hours. Changes in MCL1 protein levels were measured by reverse phase protein array. MCL1 knockdown was achieved by locked nucleic acid oligonucleotides (LNAs) and lentiviral silencing in two different prednisolone resistant leukemic cell lines, and the effect was assessed with RTQ-PCR and Western blot. Cell viability and cell count were analyzed by MACSQuant. Glucose consumption was measured using the GAGO20 glucose assay, in which glucose is oxidized to form the spectrophotometric end-product Oxidized o-Dianisidine. 2-deoxyglucose (2DG) was used to inhibit glycolysis. Cytotoxicity of prednisolone in leukemic cells was determined by the in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) drug-resistance assay. Sensitivity and resistance to prednisolone was assessed using previously established LC50 cut-off values, shown to be linked to the prognosis of patients. Results: MCL1 protein expression decreased by 2.9-fold after in vitro prednisolone treatment in prednisolone sensitive patients' leukemic cells (p<0.001). In contrast, MCL1 protein expression increased in prednisolone resistant ALL patient cells by maximum 2.3-fold (p<0.01). Three MCL1 LNA oligonucleotides efficiently diminished MCL1 protein levels by 82±16% compared to MCL1 levels measured in non-silencing control cells (p<0.05). This decrease was similar to the reduction by 72±12% seen for 2 lentivirally delivered shMCL1 (p<0.05). Silencing of MCL1 decreased leukemic cell proliferation by 9-fold and sensitized leukemic cells to prednisolone by maximum 80-fold (p<0.05). MCL1 silencing by either MCL1 LNA or shMCL1 upregulated the glucose consumption of leukemic cells by 2.5-fold (p<0.05), indicating a rescue mechanism mediated by anaerobic glycolysis. Inhibition of the anaerobic glycolysis by 2-DG diminished the proliferation rate of MCL1-silenced cells by 3.9-fold compared to MCL1-silenced cells alone (p<0.05). Most importantly, the combination of 2DG and silencing of MCL1 synergistically sensitized to prednisolone by 33±16% compared to the prednisolone response of leukemic cells treated with 2DG or MCL1 LNA alone (p<0.05, n=3). Conclusion: MCL1 is a potent target to sensitize to prednisolone in pediatric ALL. However, MCL1-silenced cells increase anaerobic glycolysis to avoid prednisolone-induced apoptosis. MCL1 and glycolysis should therefore be targeted simultaneously to effectively and synergistically reverse prednisolone resistance in ALL. Disclosures: No relevant conflicts of interest to declare.

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Hydrocortisone does Not Influence in Vitro Glucocorticoid Sensitivity of Acute Lymphoblastic Leukemia Blasts

Blood ◽

10.1182/blood.v124.21.5228.5228 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5228-5228

Author(s):

Lidewij T. Warris ◽

Marry M. van den Heuvel-Eibrink ◽

Ingrid M. Ariës ◽

Rob Pieters ◽

Erica L.T. van den Akker ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Side Effects ◽

Cell Lines ◽

Fluorescence Intensity ◽

Lymphoblastic Leukemia ◽

Leukemic Cell ◽

Leukemic Cells ◽

Glucocorticoid Sensitivity ◽

Leukemic Cell Lines

Abstract Introduction: Dexamethasone-induced neuropsychological side effects on mood, behavior and cognition seriously affect quality of life in children with acute lymphoblastic leukemia (ALL) during a long treatment period. Based on recent studies in animals, we hypothesized that these neuropsychological side effects are mediated by dexamethasone-induced cortisol depletion of the mineralocorticoid receptor (MR) in the brain. Therefore, we hypothesize that these side effects could be ameliorated by an intervention with hydrocortisone. For clinical application settings however, an absolute prerequisite is that MR activation does not interfere with the efficacy of the glucocorticoids, dexamethasone and prednisolone, on ALL cells. Materials and Methods: To investigate responsiveness of leukemic cell lines and fresh patients’ leukemic cells to dexamethasone and prednisolone in the presence of hydrocortisone, MTT-assays were performed. In addition MR and the glucocorticoid receptor (GR) expression on leukemic cells of different ALL subtypes was studied with a microarray-based gene expression profiling and validated by quantitative real-time PCR. Results: Leukemic cells expressed the MR at a very low level with a significantly higher (P≤0.001) expression in ETV6-RUNX1+ patients (median: 160.7 [AU] of fluorescence intensity, range: 38.1 - 760.6 [AU]) versus other ALL subtypes (median: 41.8 [AU] of fluorescence intensity, range: 25.1 - 276.2 [AU]). MR expression did not differ between glucocorticoid resistant and sensitive patients’ cells. Hydrocortisone addition did not affect glucocorticoid sensitivity of leukemic cell lines and patients’ leukemic cells of different leukemic subtypes also including ETV6-RUNX1+. Glucocorticoid sensitive patients’ cells became significantly more sensitive by hydrocortisone addition (prednisolone: P≤0.01, dexamethasone: P≤0.05). Conclusion: This present study shows that hydrocortisone does not interfere with efficacy of dexamethasone and prednisolone in vitro. These findings support a clinical randomized trial to study whether addition of hydrocortisone decreases the neuropsychological side effects of dexamethasone in children with ALL. Acknowledgments: The financial support of the KiKa® (Kinderen Kankervrij) foundation is highly appreciated. Disclosures No relevant conflicts of interest to declare.

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Clinical relevance of BCL-2 overexpression in childhood acute lymphoblastic leukemia

Blood ◽

10.1182/blood.v87.3.1140.bloodjournal8731140 ◽

1996 ◽

Vol 87 (3) ◽

pp. 1140-1146 ◽

Cited By ~ 145

Author(s):

E Coustan-Smith ◽

A Kitanaka ◽

CH Pui ◽

L McNinch ◽

WE Evans ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Peripheral Blood ◽

Lymphoblastic Leukemia ◽

Philadelphia Chromosome ◽

Leukemic Cell ◽

Trophic Factors ◽

Chromosomal Translocations ◽

Childhood All ◽

Protein Levels

Enforced BCL-2 gene expression in leukemic cell lines suppresses apoptosis and confers resistance to anticancer drugs, but the clinical significance of increased BCL-2 protein levels in acute lymphoblastic leukemia (ALL) is unknown. Among 52 children with newly diagnosed ALL, BCL-2 expression in leukemic lymphoblasts ranged widely, from 4,464 to 59,753 molecules of equivalent soluble fluorochrome per cell (MESF), as determined by flow cytometry. The mean (+/- SD) level of MESF in 43 cases of B-lineage ALL (19,410 +/- 11,834) was higher than that detected in CD10+ B-lymphoid progenitors from normal bone marrow (450 +/- 314; P < .001), and CD19+ peripheral blood B lymphocytes (7,617 +/- 1,731; P = .02). Levels of BCL-2 in T-ALL cases (17,909 +/- 18,691) were also generally higher than those found in normal CD1a+ thymocytes (1,762 +/- 670), or in peripheral blood T lymphocytes (9,687 +/- 3,019). Although higher levels of BCL-2 corresponded to higher leukemic cell recoveries after culture in serum-free medium, they did not correlate with higher cell recoveries after culture on stromal layers, or with in vitro resistance to vincristine, dexamethasone, 6- thioguanine, cytarabine, teniposide, daunorubicin or methotrexate. BCL- 2 protein levels did not correlate with presenting clinical features. Unexpectedly, however, lower-than-median MESF values were significantly associated with the presence of chromosomal translocations (P = .010). Notably, all six cases with the Philadelphia chromosome, a known high- risk feature, had low levels of BCL-2 expression (P = .022). Higher levels of BCL-2 were not associated with poorer responses to therapy among 33 uniformly treated patients, and were not observed in three patients studied at relapse. In conclusion, increased BCL-2 expression in childhood ALL appears to enhance the ability of lymphoblasts to survive without essential trophic factors, and is inversely related to the presence of chromosomal translocations. However, it does not reflect increased disease aggressiveness or resistance to chemotherapy.

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Colony formation in vitro by leukemic cells in acute lymphoblastic leukemia (ALL)

Blood ◽

10.1182/blood.v52.4.712.712 ◽

1978 ◽

Vol 52 (4) ◽

pp. 712-718 ◽

Cited By ~ 28

Author(s):

SD Smith ◽

EM Uyeki ◽

JT Lowman

Keyword(s):

Bone Marrow ◽

Acute Lymphoblastic Leukemia ◽

Bone Marrow Cells ◽

Lymphoblastic Leukemia ◽

Lymphoid Cells ◽

Assay System ◽

Leukemic Cells ◽

Specific Cell ◽

Specific Cell Surface

Abstract An assay system in vitro for the growth of malignant lymphoblastic colony-forming cells (CFC) was established. Growth of malignant myeloblastic CFC has been previously reported, but this is the first report of growth of malignant lymphoblastic CFC. Established assay systems in vitro have been very helpful in elucidating the control of growth and differentiation of both normal and malignant bone marrow cells. Lymphoblastic CFC were grown from the bone marrow aspirates of 20 children with acute lymphoblastic leukemia. Growth of these colonies was established on an agar assay system and maintained in the relative hypoxia (7% oxygen) of a Stulberg chamber. The criteria for malignancy of these colonies was based upon cellular cytochemical staining characteristics, the presence of specific cell surface markers, and the ability of these lymphoid cells to grow without the addition of a lymphoid mitogen. With this technique, specific nutritional requirements and drug sensitivities can be established in vitro, and these data may permit tailoring of individual antileukemic therapy.

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BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

Cancer Cell International ◽

10.1186/s12935-021-01908-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Shuiyan Wu ◽

You Jiang ◽

Yi Hong ◽

Xinran Chu ◽

Zimu Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Acute Lymphoblastic Leukemia ◽

Caspase 3 ◽

Lymphoblastic Leukemia ◽

Rna Seq ◽

Cell Acute Lymphoblastic Leukemia ◽

Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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Low molecular weight B cell growth factor induces proliferation of human B cell precursor acute lymphoblastic leukemias

Blood ◽

10.1182/blood.v70.1.132.bloodjournal701132 ◽

1987 ◽

Vol 70 (1) ◽

pp. 132-138 ◽

Cited By ~ 1

Author(s):

B Wormann ◽

SR Mehta ◽

AL Maizel ◽

TW LeBien

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Growth Factor ◽

Cell Growth ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Cell Precursor ◽

B Cell Growth Factor ◽

B Cell Precursors

Experiments were conducted to determine the effect of low mol wt B cell growth factor (L-BCGF) on B cell precursor acute lymphoblastic leukemia (ALL). L-BCGF induced a significant increase in 3H-TdR incorporation in 28 of 37 bone marrow aspirates from patients with B cell precursor ALL, with stimulation indices ranging from 2 to 129. Fluorescence-activated cell sorting confirmed that in five of seven patients the common acute lymphoblastic leukemia antigen (CALLA)/CD10 positive leukemic cells were responding directly to L-BCGF. L-BCGF was capable of inducing, in some patients, an increase in absolute viable cells and could also induce colony formation in vitro. The response of B cell precursor ALL was not attributable to beta IL 1, IL 2, or gamma interferon. These results indicate that the majority of B cell precursor ALL undergo a proliferative response to L-BCGF, suggesting a regulatory role for this lymphokine in the growth of B cell precursors.

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