Preclinical Investigation of the p53-MDM2 Antagonist Idasanutlin (RG7388) Demonstrates Significant Activity in High Risk Adult Acute Lymphoblastic Leukemia

Outcomes for adult patients with acute lymphoblastic leukemia (ALL) are dismal and have not kept pace with their pediatric counterparts, with five-year survival rates of less than 45%. TP53 mutations are infrequent in ALL, but activity of the oncoprotein MDM2 may otherwise phenotypically disrupt and circumvent normal p53 function, positing the p53-MDM2 signaling axis as a potential therapeutic target for the engagement of intrinsic cell death programs. Given the clinical safety and responses to p53-MDM2 antagonist idasanutlin (RG7388) in other hematological cancers and solid tumors, we aimed to evaluate the therapeutic potential of idasanutlin in ALL. Single-agent activity of idasanutlin was investigated in 17 high-risk ALL patient and patient-derived xenograft samples (aged 4 to 51 years), both B-ALL (n=15) and T-ALL (n=2), including KMT2A-rearranged, TCF3-rearranged, and Philadelphia-positive ALL. An ex vivo coculture of ALL blasts and hTERT-immortalized mesenchymal stem cells (MSC) was employed to support growth of the ALL blasts during short-term culture, complemented by a fluorescent image-based microscopy platform which identifies and discriminates the two cellular compartments using random forest machine learning algorithms based on cellular nuclear staining. Idasanutlin demonstrated sub-micromolar, dose-dependent anti-leukemic activity against 15 of 17 samples tested, with half maximal effective concentrations (EC50) in the range of 10 to 220 nM (mean EC50 = 45.1nM); the two exceptions were later determined to harbor homozygous inactivating TP53 mutations; p.Y220C and p.S241P within the p53 DNA binding domain. The idasanutlin EC50 concentrations determined are clinically achievable, well below the peak plasma concentrations reported in patients for other disease indications. Furthermore, idasanutlin concentrations below 10µM had no impact upon MSC survival. Conforming to p53-MDM2 auto-regulatory feedback mechanisms, we demonstrated that idasanutlin efficiently stabilized and activated p53 at the protein level within 6 hours when treated with their respective idasanutlin EC50, to a level greater than 4-fold increased relative to their respective vehicle-only controls (p=0.001, n=7). Further, p53-regulated transcriptional target gene products, MDM2 and p21, were increased by 5-fold and 2-fold respectively, validating engagement of the p53 pathway by idasanutlin (p=0.036 and 0.125, respectively). By contrast, idasanutlin did not elicit increased expression of either p53-regulated transcriptional target gene product in the identified TP53-mutant patient samples (n=2). On-target specificity of idasanutlin was further confirmed in a NALM6 isogenic cell line model, whereby the TP53 wildtype line was sensitive to idasanutlin (EC50 = 74nM) and effectively activated p53 signaling whereas the TP53 homozygous null line was highly resistant (EC50 = ~10µM). To determine whether the decreased cell numbers and engagement of p53 signaling observed were accompanied by cell death, the capacity of idasanutlin to induce apoptosis in the ALL samples was next investigated. Patient-derived ALL samples (n=6) were treated with vehicle or idasanutlin at their respective EC50s for 24 and 48 hours, and then analyzed by flow cytometry. There was an increase in annexin-V positive cells within 24 hours compared to the vehicle-only treated cells (mean±SD 14.3±6.6% vs 27.0±21.2% respectively (p=0.125). By 48 hours significant apoptosis was attained, with a mean±SD of 59.0±23.8% annexin-V positive cells compared to the mean of vehicle-treated cells at 29.1±11.6% (p=0.004). Cleaved poly(ADP-ribose) polymerase (PARP) levels were also increased greater than 3-fold compared to vehicle-only control cells as assessed by immunoblotting (p=0.045, n=3), corroborating these findings. These data emphasize the potential of pharmacologically targeting the p53-MDM2 axis in ALL, demonstrating potent, on-target, cytotoxic activity in a range of high-risk ALL cytogenetic subgroups. Taken together, these findings support further preclinical investigations into idasanutlin and other p53-MDM2 antagonists and potential combinations to improve the treatment of adult ALL. Disclosures Irving: F. Hoffmann-La Roche: Research Funding.

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Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia

Blood ◽

10.1182/blood.v83.7.1731.1731 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1731-1737 ◽

Cited By ~ 54

Author(s):

A Manabe ◽

E Coustan-Smith ◽

M Kumagai ◽

FG Behm ◽

SC Raimondi ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Programmed Cell Death ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Fatal Outcome ◽

Interleukin 4 ◽

B Lineage

Abstract We investigated the effects of interleukin-4 (IL-4) on the survival of leukemic and normal B-cell progenitors cultured on bone marrow stroma. IL-4 (at 100 U/mL) was cytotoxic in 16 of 21 cases of B-lineage acute lymphoblastic leukemia, causing reductions in CD19+ cell numbers that ranged from 50% to greater than 99% (median 83.5%) of those in parallel cultures not exposed to the cytokine. All nine cases with the t(9;22)(q34;q11) or the t(4;11)(q21;q23), chromosomal features that are often associated with multidrug resistance and a fatal outcome, were susceptible to IL-4 toxicity. IL-4 cytotoxicity resulted from induction of programmed cell death (apoptosis); there was no evidence of cell killing mediated by T, natural killer, or stromal cells. IL-4 cytotoxicity extended to a proportion of normal B-cell progenitors. After 7 days of culture with IL-4 at 100 U/mL, fewer CD19+, CD34+ normal lymphoblasts (the most immature subset) survived: in five experiments the mean (+/- SEM) reduction in cell recoveries caused by IL-4 was 60.0% +/- 6.0%. By contrast, reductions in recovery of more differentiated bone marrow B cells (CD19+, CD34-, surface Ig+) were low (6.6% +/- 2.2%; P < .001 by t-test). Our findings indicate that IL-4 is cytotoxic for human B-cell precursors and support clinical testing of IL-4 in cases of high-risk lymphoblastic leukemia resistant to conventional therapy.

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

Blood ◽

10.1182/blood.v116.21.1809.1809 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1809-1809

Author(s):

Hisashi Harada ◽

Nastaran Heidari ◽

Mark Hicks

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Small Molecule ◽

Caspase 3 ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Agent ◽

Annexin V ◽

Glucocorticoid Resistance ◽

Down Regulation

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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Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.903.903 ◽

2015 ◽

Vol 126 (23) ◽

pp. 903-903

Author(s):

Salih Demir ◽

Galina Selivanova ◽

Eugen Tausch ◽

Lisa Wiesmüller ◽

Stephan Stilgenbauer ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Cell Lines ◽

Therapeutic Intervention ◽

High Performance ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Therapy Resistance ◽

Leukemia Cells

Abstract Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53 has so far not been addressed as a target for therapeutic intervention. In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) orwild type (wt) TP53. Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53 mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53 mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53 mut and 4 TP53 wt lines. Exposure of BCP-ALL primograft (TP53 mut n=4, TP53 wt n=4) and cell line (TP53 mut n=2, TP53 wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53 mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53 mut 49 and 143 ng/ml, TP53 wt mean 12 ng/ml) and lower induction of cell death (TP53 mut 16 to 23%, TP53 wt 10 to 60%) in TP53 mut ALL, corroborating the tumor-suppressive function of p53 in ALL. We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53 mut (IC50: 5 µM for both cell lines) as compared to TP53 wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53 mut but not TP53 wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53 mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53 mut compared to no activation in TP53 wt leukemia cells, indicating restoration of p53function upon APR-246treatment in BCP-ALL. Based on these findings, we addressed the effectivity of APR-246on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53 mut (n=4) and TP53 wt (n=4) samples. Importantly, the pattern of responsiveness of TP53 mut ALL was also identified in TP53 mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53 mut ALL (TP53 mut 48%, TP53 wt 18%, 5 µM APR-246, 24 h). Taken together, we showed that TP53 mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL. Disclosures Selivanova: Aprea: Patents & Royalties: APR-246. Tausch:Gilead: Other: Travel support. Stilgenbauer:Gilead: Honoraria, Research Funding.

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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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Interleukin-4 induces programmed cell death (apoptosis) in cases of high-risk acute lymphoblastic leukemia

Blood ◽

10.1182/blood.v83.7.1731.bloodjournal8371731 ◽

1994 ◽

Vol 83 (7) ◽

pp. 1731-1737 ◽

Cited By ~ 2

Author(s):

A Manabe ◽

E Coustan-Smith ◽

M Kumagai ◽

FG Behm ◽

SC Raimondi ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Programmed Cell Death ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Fatal Outcome ◽

Interleukin 4 ◽

B Lineage

We investigated the effects of interleukin-4 (IL-4) on the survival of leukemic and normal B-cell progenitors cultured on bone marrow stroma. IL-4 (at 100 U/mL) was cytotoxic in 16 of 21 cases of B-lineage acute lymphoblastic leukemia, causing reductions in CD19+ cell numbers that ranged from 50% to greater than 99% (median 83.5%) of those in parallel cultures not exposed to the cytokine. All nine cases with the t(9;22)(q34;q11) or the t(4;11)(q21;q23), chromosomal features that are often associated with multidrug resistance and a fatal outcome, were susceptible to IL-4 toxicity. IL-4 cytotoxicity resulted from induction of programmed cell death (apoptosis); there was no evidence of cell killing mediated by T, natural killer, or stromal cells. IL-4 cytotoxicity extended to a proportion of normal B-cell progenitors. After 7 days of culture with IL-4 at 100 U/mL, fewer CD19+, CD34+ normal lymphoblasts (the most immature subset) survived: in five experiments the mean (+/- SEM) reduction in cell recoveries caused by IL-4 was 60.0% +/- 6.0%. By contrast, reductions in recovery of more differentiated bone marrow B cells (CD19+, CD34-, surface Ig+) were low (6.6% +/- 2.2%; P < .001 by t-test). Our findings indicate that IL-4 is cytotoxic for human B-cell precursors and support clinical testing of IL-4 in cases of high-risk lymphoblastic leukemia resistant to conventional therapy.

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

Blood ◽

10.1182/blood-2018-99-117139 ◽

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.

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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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Autophagy Inhibition Sensitizes Acute Lymphoblastic Leukemia Cells to L-Asparaginase

Blood ◽

10.1182/blood.v126.23.3772.3772 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3772-3772 ◽

Cited By ~ 1

Author(s):

Hiroyoshi Takahashi ◽

Jun Inoue ◽

Kimiyoshi Sakaguchi ◽

Masatoshi Takagi ◽

Shuki Mizutani ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Combination Treatment ◽

Lymphoblastic Leukemia ◽

Annexin V ◽

Leukemia Cells ◽

G1 Phase ◽

Autophagy Inhibition

Abstract Autophagy is an intracellular protein and organelle degradation system, and is upregulated in response to cellular stress such as amino acid starvation. On the other hand, L-asparaginase (L-asp) plays an essential role in acute lymphoblastic leukemia (ALL) therapy by reducing intracellular asparagine and glutamine in ALL cells. However, the relationship of L-asp and autophagy is largely unknown. Here we show that L-asp induced cytoprotective autophagy. Three ALL cell lines of varied genetic background were used for in vitro experiments (REH, ETV6-RUNX1+ B-cell precursor (BCP) ALL; 697, E2A-PBX1+ BCP-ALL; TS-2, MEF2D-DAZAP1+ BCP-ALL). The cells were exposed to chroloquine (CQ) or bafilomycin A1 as autophagy inhibitors for 3 hours. LC3B-II, autophagy flux marker, was significantly increased under L-asp treatment with CQ as compared to only CQ condition, which was confirmed in independent experiments at immunofluorescence staining. Transmission electron microscopy showed that both the number and the area of autophagic vesicles per cell were markedly increased in L-asp with CQ condition. Thus, autophagy was induced by L-asp increasing turnover and clearance of autophagosomes in ALL cells. The toxic effect of 4 groups (control, CQ, L-asp, and L-asp plus CQ) by flow cytometry using Annexin-V staining indicated that combination treatment with L-asp and CQ for 48 hours induced significant cell death in the three ALL cell lines. Furthermore, inhibition of autophagy by CQ comparably sensitize REH cells to L-asp as ATG7 silencing by short interfering RNA. Cell growth assays for 6-9 days showed that L-asp monotreatment suppressed cell growth but did not increase the percentage of dead cells. In contrast, combination treatment with L-asp and CQ decreased the number of living cells and significantly increased the percentage of dead cells in time-dependent manner. Cell cycle analysis showed that cell cycle arrest at G1 phase was induced and the percentage of cells in sub-G1 phase remained a small increase by L-asp monotreatment, indicating leukemia cells endured amino acid deficiency by G1 arrest. In contrast, addition of CQ to L-asp significantly increased the sub-G1 population instead of decreasing G1 population. The apoptosis-related protein expressions using western blot analysis showed that combination treatment with L-asp and CQ induced cleavage of caspase 3 and PERP. In addition, a pan-caspase inhibitor benyloxycarbonyl Val-Ala-Asp (O-methyl)-fluoro-methylketone (z-VAD) significantly reduced the percentage of Annexin-V positive cells in the combination treatment with L-asp and CQ, which suggested that the autophagy inhibition upon L-asp treatment induced apoptotic cell death. We next transduced REH cell line with a luciferase-expressing viral vector. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice were transplanted with these cells via tail vein and 6,000 U/kg L-asp and/or 50 mg/kg CQ were injected intraperitoneally once per day for survival analysis. The combination treatment with L-asp and CQ clearly reduced the leukemia burden as detected by luciferase intensity and improved outcome (L-asp plus CQ vs L-asp at day 28 after administration: 82% vs 0%, respectively. p <0.001). Of note, LC3B dots detected by immunofluorescence staining were apparently increased by the combination treatment with L-asp and CQ in the ALL cells derived from peripheral blood, bone marrow, and central nervous system at day 14 after transplantation. Taken together, these data suggest that autophagy plays cytoprotective role in L-asp-treated ALL cells both in vitro and in vivo, and autophagy inhibition upon L-asp treatment may be a useful approach for ALL. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v128.22.4020.4020 ◽

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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The Bioflavinoid Quercetin Induces Apoptosis and Inhibits Autophagy in Primary Acute Lymphoblastic Leukemia in-Vitro

Blood ◽

10.1182/blood.v128.22.5148.5148 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5148-5148

Author(s):

John Mariano ◽

Liana M Toia ◽

Jessica C Shand

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

High Risk ◽

Western Blot ◽

Lymphoblastic Leukemia ◽

Cellular Stress ◽

Substantial Reduction ◽

Beclin 1 ◽

Standard Risk ◽

Quercetin Treatment

Abstract Quercetin is an antioxidant previously shown to inhibit acute lymphoblastic leukemia (ALL) growth and induce both apoptosis and autophagy in malignant hematologic cell lines derived from AML and T-ALL. Since autophagy is a critical mechanism in the response to cellular stress, we hypothesized that high-risk ALL cells had higher resting autophagy than standard risk ALL cells. To test our hypothesis, we quercetin-treated stable cell cultures of ALL isolated from the diagnostic marrow specimens of one clinically high-risk (HR-ALL, an infant carrying the t(4;11) MLL-rearrangement) and one cytogenetically normal standard-risk pediatric patient (SR-ALL), and compared viability and the expression of molecular markers of programmed cell death and autophagy by Western Blot. First, we established a dose-response curve that identified 50 uM quercetin as the target dose for potency and efficacy in HR- and SR-ALL. Next, we compared viability between HR-and SR-ALL treated with 50 uM quercetin at 3, 12, 24 and 48 hours. HR-ALL treated with quercetin exhibited a significantly greater reduction in viability measured by Annexin V/PI staining and MTT reagent conversion (2-way ANOVA p = 0.04 and p = 0.005). HR-ALL cells also exhibited greater catalytic cleavage and activation programmed death markers, Caspase 3 and PARP1 by Western Blot, compared to SR-ALL. Because high levels of autophagy can induce cell death, we asked whether HR-ALL cells had a higher resting autophagy rate. We compared expression of the prototypic autophagy protein, Beclin-1 at 30min, 1hr, 2hr, 3hr, and 4hr after treatment with 10mM of the autophagy inhibitor 3-methyladenine (3-MA). Because 3-MA inhibits autophagy at the early stage of nucleation, the rate of breakdown of downstream proteins such as Beclin-1 is a proxy measure for the rate of resting, non-stress induced autophagy. HR-ALL cells exhibited a significantly greater rate of Beclin-1 breakdown after 3-MA treatment as compared to SR-ALL cells (slope -0.235 vs. 0.131, p = 0.003) consistent with a higher resting autophagy rate in HR-ALL. To determine whether quercetin treatment impacts this higher rate of autophagy, we treated HR-ALL and SR-ALL cells with 50uM of quercetin for 24hrs and compared, by Western blot, the expression of regulatory proteins at each stage of autophagy: nucleation (ATG7, Beclin-1), expansion (ATG5, ATG16L1) and maturation (LC3A/B). Unexpectedly, quercetin inhibited expression of all autophagy proteins, with a greater decrease in HR-ALL. We next parsed whether selective autophagy inhibition at formation of the autophagosome, using 3MA, or later endosome acidification, using chloroquine, could explain the differential cytotoxicity of quercetin in HR-ALL. 3MA and chloroquine treatment alone resulted in significantly fewer Annexin+/PI+ HR- and SR-ALL cells and less MTT conversation compared to quercetin treatment alone (p<0.05) suggesting that selective inhibition of the autophagy complex was insufficient to explain quercetin cytotoxicity. Finally, we asked whether differences in an upstream regulator of the autophagy/apoptotic balance contribute to higher quercetin toxicity in HR-ALL cells. The regulatory protein high mobility group box-1 (HMGB1) has been shown to induce autophagy by interacting with Beclin-1, and we have previously demonstrated overexpression of HMGB1 in HR-ALL. We verified interaction of HMGB1 with Beclin-1 by immunoprecipitation, and observed a substantial reduction in complex formation after 50 uM quercetin treatment in HR-ALL cells. (1511153 vs. 70442 Beclin-1 band volumes after IP p<0.001), along with decreased expression of the anti-apoptotic proteins, Bcl-2 and Mcl-1, factors proposed to be regulated by the Beclin-1/HMGB1 complex. Taken together, these data indicate that regulation of the autophagy-apoptotic balance is a mechanism used by ALL cells to mitigate cellular stress, and is the first to describe the effects of quercetin in the context of autophagy in ALL. Future studies will compare the expression profile of critical autophagy proteins following chemotherapy treatment in a larger cohort of high-risk ALL cells, and delineate the role of HMGB1 as an autophagy regulator using our established knockdown model. Disclosures No relevant conflicts of interest to declare.

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