Evaluation Of Sphingosine Kinase 1 As a Therapeutic Target In B-Lineage Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1426-1426
Author(s):  
Craig T Wallington-Beddoe ◽  
Stuart M Pitson ◽  
Jason A Powell ◽  
Kenneth F Bradstock ◽  
Linda J Bendall
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Parp Cleavage ◽  
Signalling Pathways ◽  
Wide Range ◽  
Ic50 Values

Abstract Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over-expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. Furthermore, SphK1 has been shown to correlate with the clinical outcome of certain tumors. Here we focus on SphK1 as an important oncogenic target in acute lymphoblastic leukemia (ALL). We have previously shown SphK1 protein to be over-expressed and activated (Ser225 phosphorylation) in ALL cell lines and primary patient samples compared to normal CD34+CD19+ B-cell progenitors. Furthermore, we have reported the importance of SphK1 in the development of ALL by transducing B-cell progenitors isolated from WT or SphK1-/- mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated WT mice. The absence of SphK1 significantly reduced the incidence of ALL in recipient mice (ASH 2012). Inhibition of SphK1 by the selective inhibitor SK1-I significantly reduced intra-cellular S1P concentrations (p=0.017 and p=0.003 at 24 and 48 hours respectively) in 3 cell lines examined, indicating that the drug targets this enzyme. SK1-I killed ALL cells as determined by annexin V/PI flow cytometric analysis with IC50 values ranging from 12 µM to 18 µM at 72 hours. Furthermore, SK1-I induced cell death in primary patient ALL cells by 16 hours. This agent resulted in virtually no caspase-3 cleavage and cell death was not prevented by the pan-caspase inhibitor Z-VAD-FMK (p=0.45, n=4). Marked cytoplasmic vacuolation was detected by light microscopy, with LC3 processing present by Western blot, consistent with the development of autophagy. However, the autophagy inhibitor 3MA failed to prevent SK1-I-mediated cell death. These results suggest that the cell death associated with inhibition of SphK1 in ALL cells is caspase-independent and cannot be attributed to autophagy. Surprisingly, conventional chemotherapeutic agents such as doxorubicin and vincristine failed to synergize with SK1-I, however, synergistic killing was observed when SK1-I was combined with 500 nM imatinib over 72 hours in Philadelphia-positive (BCR/ABL+) ALL cells. We have developed a novel SphK1 inhibitor, MP8, that targets the enzyme via a different mechanism to SK1-I, since it blocks ATP binding. MP8 reduced intra-cellular S1P in Jurkat cells by 43% compared to untreated controls, and killed Jurkat and SUP-B15 cells over 24 to 48 hours with IC50 values of 8 µM. Additionally, MP8 induced cell death in primary patient ALL cells by 24 hours. This agent resulted in classic apoptotic cell death, which was rescued by Bcl-2 over-expression, resulting in near complete reversal of PARP cleavage. SphK1 has indisputable tumor-promoting properties and lies downstream of a number of signalling pathways known to be dysregulated in ALL. Here we show that SphK1 is over-expressed and activated in ALL cells and targeting SphK1 has potent cytotoxic effects in a wide range of cell lines and patient samples. Furthermore, genetic deletion of Sphk1 significantly reduced the incidence of murine BCR/ABL-driven ALL. These findings suggest further examination of the role SphK1 plays in ALL will uncover novel interactions with oncogenic signalling pathways and paves the way for the inclusion of SphK1 inhibitors in future pre-clinical trials. Disclosures: No relevant conflicts of interest to declare.

FTY720 Has Potent Anti-Leukemic Effects on Acute Lymphoblastic Leukemia Cells and Results In Caspase Independent Cell Death

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3260-3260
Author(s):  
Craig T. Wallington-Beddoe ◽  
John Hewson ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Cell Lines ◽  
Western Blotting ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Ic50 Values

Abstract Abstract 3260 Introduction: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. FTY720 is an immunosuppressive drug that has recently demonstrated efficacy in phase 3 trials of relapsing/remitting multiple sclerosis. FTY720 also appears promising in a number of malignancies with the proposed mechanism being the reactivation of PP2A, a protein serine/threonine phosphatase whose activity may be reduced in malignant cells. Here we report findings of in vitro testing of FTY720 on Ph+ and negative ALL cell lines and primary patient samples, describing mechanisms of cell death. Methods: ALL cell lines and primary patient samples were treated with 1 nM - 100 μM FTY720 for 24 hours. Viability was measured by flow cytometry using propidium iodide and annexin V staining. Cellular proliferation was measured by 3H-thymidine incorporation. Flow cytometry and western blotting were used to measure caspase 3 activation whilst western blotting was used to assess caspase 3, PARP cleavage and LC3II formation. Electron microscopy permitted a detailed examination of cell ultra-structure and confocal microscopy with lysosensor blue staining enabled visualisation of acidic vacuoles. Reactive oxygen species generation was assessed by flow cytometry using the cell permeable dye carboxy-H2DCFDA. Results: FTY720 produced a profound reduction in proliferation and viability of Ph+ (ALL1 cells) and Ph− (REH, NALM6 and LK63 cells) cell lines and patient samples (n=7) in the low micromolar range. IC50 values for loss of viability at 24 hours ranged from 5.3 μM for ALL1 to 7.9 μM for LK63. The IC50 values for proliferation at 24 hours were 1.4 μM for ALL1 and 3.5 μM for REH. Caspase 3 activation was observed only at very low levels by flow cytometry whilst both caspase 3 and PARP cleavage were not detected by western blotting. Inhibition of caspases by ZVAD-FMK failed to rescue ALL cells from FTY720 induced cell death, demonstrating a caspase independent cell death mechanism. Light microscopy revealed prominent cytoplasmic vacuolation, and electron microscopy showed features consistent with autophagy and necrosis. Western blotting demonstrated strong LC3II bands and confocal microscopy, using lysosensor blue, revealed prominent acidic vacuolation, all confirming the induction of autophagy. Reactive oxygen species were generated in response to FTY720 treatment and partial reversal of this by N-acetyl-cysteine produced a concomitant increase in cell viability. PP2A inhibition with okadaic acid failed to rescue cells from FTY720-induced cell death. Conclusion: FTY720 is a highly active drug in vitro in ALL cell lines and patient samples. Evidence supports a caspase independent mechanism of cell death with the occurrence of autophagy and necrosis. PP2A activation is not solely responsible for leukemic cell death. Data on the in vivo effects of FTY720 on ALL cells in NOD-SCID mice will be presented. Disclosures: Bendall: Genzyme: Honoraria.

scholarly journals AT1412, a Patient-Derived Antibody in Development for the Treatment of CD9 Positive Precursor B-Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4461-4461
Author(s):  
Greta De Jong ◽  
Sophie E Levie ◽  
Remko Schotte ◽  
Wouter Pos ◽  
Daniel Go ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Melanoma Cells ◽  
Employment Equity ◽  
Equity Ownership

Despite rapid advances in immunotherapeutic options for precursor B-acute lymphoblastic leukemia (ALL), outcomes remain poor especially for adult ALL and relapsed pediatric ALL. With conventional chemotherapy, remission percentages in adult ALL range from 75 to 90%, but relapse rates are high and long-term leukemia-free survival ranges between 35-70% depending on age and risk group. The introduction of CD19 targeting immunotherapy has significantly improved patient outcomes in (relapsed) B-ALL. However, tumor escape via downregulation of CD19 occurs in a significant number of patients. Therefore an ongoing urgency remains for the identification of additional or alternative immunotherapeutic targets for the treatment of ALL. AT1412 is an antibody that was identified from the peripheral blood memory B cell pool of a patient cured of metastatic melanoma after adoptive T-cell therapy, using a B cell immortalization technology (AIMSelect) with ectopic Bcl-6 and Bcl-xL expression as described previously [Kwakkenbos et al. Nat. Med. 2010]. The antibody was selected based on differential binding to melanoma cells as compared to healthy melanocytes and was shown to be successful in killing melanoma cells in vitro and in vivo [manuscript submitted]. In addition to melanoma, AT1412 binds other tumor types including B-ALL, gastric, colon- and pancreatic cancer. The target of AT1412 is the tetraspanin CD9, which is expressed by more than half of all B-ALL. Expression of CD9 has been correlated with adverse prognosis [Liang et al. Cancer Biomark. 2018]. We assessed binding of this human CD9 antibody to a panel of ALL cell lines using flow cytometry. Binding of AT1412 to the B-ALL cell lines SUP-B15, MHH-CALL-2 and CCRF-SB varied as expected based on the CD9 levels that we detected using a commercial CD9 antibody. AT1412 induced antibody dependent cellular cytotoxicity (ADCC) on these cells, in line with the level of AT1412 binding. No binding was seen to the T-ALL cell line Jurkat. Importantly, these findings were confirmed in primary ALL samples, obtained prospectively at diagnosis from a cohort of patients with T- or B-ALL (n=30). AT1412 showed binding to 61% of B-ALL samples but not to T-ALL samples. The potential of AT1412 to induce ADCC was tested on patient samples from the same panel. Remarkably, AT1412 induced ADCC of all B-ALL samples it bound to (8 out of 14) and of none of the T-ALL samples. Cytotoxicity significantly correlated with the level of AT1412 binding. These findings were supported by the observation that AT1412 induced B-ALL cell death when a freshly drawn whole bone marrow sample from a patient with newly diagnosed B-ALL was cocultured with AT1412. AT1412-induced cell death of B-ALL blasts occurred without affecting the monocytic, granulocytic and lymphocytic populations. This cell death was not observed when this patient's ALL blasts were incubated with AML-targeting antibodies. Remarkably, AT1412 induced cell death in the absence of added effector cells or other (chemo)therapeutic agents, while the bone marrow sample contained over 80% blasts and as little as 3% lymphocytes. We are currently investigating the in vivo efficacy of the antibody in a humanized immune system mouse model with human B-ALL. Taken together, the majority of precursor B-ALL blasts express CD9 and expression of CD9 is associated with a dismal outcome. Our data demonstrate that CD9 can be successfully targeted by the human CD9 antibody AT1412, suggesting that AT1412 has the potential to be developed as a therapeutic antibody for B-ALL. AT1412 is currently being advanced through preclinical development. Disclosures De Jong: AIMM Therapeutics: Employment. Levie:AIMM Therapeutics: Employment. Schotte:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Pos:AIMM Therapeutics: Patents & Royalties: Patent WO2017119811A1. Go:AIMM Therapeutics: Employment, Patents & Royalties: Patent WO2017119811A1. Yasuda:AIMM Therapeutics: Employment, Equity Ownership. Cercel:AIMM Therapeutics: Employment. van Hal-van Veen:AIMM Therapeutics: Employment. Frankin:AIMM Therapeutics: Employment. Villaudy:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Helden:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. van Eenennaam:AIMM Therapeutics: Employment. Spits:AIMM Therapeutics: Employment, Equity Ownership, Patents & Royalties: Patent WO2017119811A1. Hazenberg:AIMM Therapeutics: Other: Employment/equity of partner/spouse.

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.

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

Blood ◽  
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.

scholarly journals Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

2020 ◽  
Author(s):  
MINORI TAMAI ◽  
Meixian Huang ◽  
Keiko Kagami ◽  
Masako Abe ◽  
Tamao Shinohara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Risk Allele ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Nucleotide Polymorphisms ◽  
Cell Precursor ◽  
Single Nucleotide ◽  
Ic50 Values

Abstract Background The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. Methods We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. Results No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p=0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p=0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). Conclusion These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

scholarly journals The NEDD8 Activating Enzyme Inhibitor Pevonedistat Induces ER Stress/UPR-Mediated Cell Death and Rebalances Homeostasis of Pro- and Anti-Apoptotic Proteins in Favor of Cell Death in Acute Lymphoblastic Leukemia Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1327-1327
Author(s):  
Gilles M. Leclerc ◽  
Shuhua Zheng ◽  
Guy J. Leclerc ◽  
Joanna DeSalvo ◽  
Ronan T. Swords ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Synthesis ◽  
Acute Lymphoblastic Leukemia ◽  
Er Stress ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Current Therapy ◽  
Parp Cleavage ◽  
E3 Ligases ◽  
Apoptotic Proteins

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children. Most adults will die from the disease. Hence, current therapy in ALL is inadequate. We previously showed that ALL cells were differentially sensitive to agents inducing endoplasmic reticulum (ER) stress (Mol Cancer Res 2012; 10:969-978; PLoS One 2013; 8:e74420). In this regard, we investigated the anti-leukemic activity of the NEDD8-activating enzyme (NAE) inhibitor pevonedistat (MLN4924). Pevonedistat prevents neddylation of cullin-RING E3 ligases, leading to accumulation of cullin dependent substrates with anti-proliferative effects. We demonstrated that pevonedistat induced dose-dependent cell growth inhibition (IC50 between 159-300 nM) and cell death (EC50 between 317-463 nM) in both T- and B-ALL cells (cell lines and primary patient material). We found that neddylated cullin levels were significantly decreased confirming inhibition of the NEDD8 conjugation pathway by pevonedistat. Pevonedistat induced ER stress and unfolded protein response (UPR) mediated cell death in ALL cell lines and primary cells as evidenced by increased expression of UPR markers (GRP78 and CHOP), and cleaved-PARP. Mechanistically, pevonedistat caused proteotoxic/ER stress from failure to halt protein translation mediated by p-eIF2α (Ser51) de-phosphorylation via up-regulation of the PERK inhibitor p58IPK. We also observed up-regulation of mTOR/p70S6K, further increasing protein synthesis and augmenting proteotoxic/ER stress. Indeed, we demonstrated that pevonedistat induced nascent protein synthesis, and that co-treatment with protein synthesis inhibitors (rapamycin, cycloheximide) rescued ALL cells from pevonedistat induced cytotoxicity (p < 0.01 for combination treatment vs. pevonedistat alone). The observed rescue correlated with decreased UPR markers and P-p70S6K (Thr389) expression, suggesting that proteotoxic stress is a central determinant in pevonedistat-induced ALL cell death. Additional studies into the mechanism of pevonedistat-induced apoptosis revealed that homeostasis of pro- and anti-apoptotic proteins was rebalanced in favor of cell death through decreased Mcl-1 pro-survival activity, via sequestration by NOXA and BIM. Co-immunoprecipitation experiments showed that pevonedistat increased the interaction between NOXA and Mcl-1, and BIM and Mcl-1 proteins, resulting in the reduction of Mcl-1 pro-survival activity. siRNA-mediated down-regulation of NOXA and BIM expression decreased the sensitivity of ALL cells to pevonedistat, supporting the sequestration by NOXA and BIM of Mcl-1's pro-survival activity. Activation of the MEK/ERK/Mcl-1 pathway following pevonedistat-induced cell death was also noted, possibly as a compensatory mechanism. We found that pevonedistat plus a MEK inhibitor (PD98059, selumetinib) induced significant cell death compared to each drug alone (p < 0.0001), which was associated with decreased Mcl-1 expression and increased cleaved-PARP cleavage, supporting a critical role of Mcl-1 in ALL cell survival. In addition, we demonstrated synergy between pevonedistat and effective anti-leukemic agents such as dexamethasone, doxorubicin and cytarabine in ALL cell line models, with CI values of 0.17, 0.46, and 0.23, respectively. More importantly, we showed that NSG mice engrafted with human ALL cells had statistically significant increased survival when treated with pevonedistat (pevo) plus dexamethasone (dex) compared to single agent therapy (p = 0.0076 for pevo+dex vs. dex alone; p = 0.0182 pevo+dex vs. pevo alone), lending support for the use of pevonedistat as part of a multi-agent approach. Taken together, our data demonstrate that the NAE inhibitor pevonedistat alters cellular translational machinery leading to ER stress/UPR-mediated cell death, and suggest that pevonedistat may have a "priming" effect on ALL cells by altering the apoptotic threshold through modulation of Mcl-1's pro-survival activity. Disclosures No relevant conflicts of interest to declare.

scholarly journals Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

2020 ◽  
Author(s):  
MINORI TAMAI ◽  
Meixian Huang ◽  
Keiko Kagami ◽  
Masako Abe ◽  
Shinpei Somazu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Risk Allele ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Nucleotide Polymorphisms ◽  
Cell Precursor ◽  
Single Nucleotide ◽  
Ic50 Values

Abstract Background The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. Methods We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. Results No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p=0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p=0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). Conclusion These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

Sphingosine Kinase Inhibition Has Pre-Clinical Activity in Acute Lymphoblastic Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3573-3573
Author(s):  
Craig T. Wallington-Beddoe ◽  
David Ho ◽  
Kenneth Francis Bradstock ◽  
Linda J. Bendall
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Term Survival ◽  
Intracellular Location ◽  
Ic50 Values

Abstract Abstract 3573 Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer, which usually responds to chemotherapy. Long-term survival in adults is poor with most developing disease relapse, whilst Ph+ ALL has a particularly poor prognosis. Sphingosine 1-phosphate (S1P) is a lipid mediator of diverse cellular functions, most notably of lymphocyte trafficking, angiogenesis, cell proliferation and survival. S1P is produced intracellularly by the sphingosine kinases (SK) of which there are two isoforms, SK1 and SK2. SK1 is over expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. The role of SK2 is much less well defined and appears to be dependent on its intracellular location with some reports of opposite effects to those of SK1. Inhibition of either SK1 or SK2 is currently under investigation as a novel anti-cancer strategy and potent anti-leukemic effects are likely. Application of the combined SK1/SK2 inhibitor SKI II and the selective SK2 inhibitor ABC294640 to ALL cells produced a reduction in cellular proliferation as measured by 3H-thymidine incorporation in all cell lines (REH, NALM6, LK63, ALL1, 2070 and TOM1) tested with IC50 values of 1μM – 7μM for SKI II and <40μM for ABC294640. Viability, measured by flow cytometry using annexin V and propidium iodide (PI) staining, was also reduced in all cell lines except the Ph+ ALL1 and 2070 cells treated with SKI II with IC50 values ranging from 2μM to >10μM for SKI II and 50–60μM for ABC294640. SKI II resulted in caspase-dependent cell death, as determined by flow cytometric assessment of intracellular caspase-3 cleavage and apoptotic morphology on light microscopy, with cell death prevented by pre-incubation with 100μM of the pan-caspase inhibitor Z-VAD-FMK. However, ABC294640 induced caspase-3 cleavage at lower than expected levels and cell death was not prevented by Z-VAD-FMK. Both agents significantly reduced intracellular S1P concentrations by 24 hours as determined by ELISA, thereby confirming the ability of these compounds to inhibit SK1 or SK2 activity. A search for agents that synergized with the SK inhibitors revealed that when Ph+ ALL cells were treated with the combination of imatinib and either ABC294640 or SKI II, a further reduction in cell death occurred than with either agent alone, thereby enhancing the therapeutic effect of ABC294640 and overcoming resistance seen with SKI II alone. Furthermore, the combination of mildly cytotoxic concentrations of ABC294640 and the novel pan histone deacetylase inhibitor AR-42 were found to significantly increase leukemic cell death at 24 and 48 hours in Ph+ and negative ALL cells. In vivo assessment of the SK inhibitors was determined by injecting NOD/SCID IL2gc−/− mice with 2–5 million human B-ALL cells and treating with 100 mg/kg/day ABC294640 or vehicle by intraperitoneal injection for 21 days after which all animals were sacrificed. Assessment of leukemia in blood, bone marrow and spleen was determined by flow cytometry using antibodies to human CD19 and murine CD45. Significant reductions in the levels of leukemia in all examined tissues were found in ABC294640-treated animals using three different human ALL xenografts, including the Ph+ positive xenograft 2070. Average absolute levels of leukemia in the bone marrow of ABC294640-treated mice for xenografts 2070, 1345 and 0398 were reduced by 40% (p = 0.00007), 55% (p = 0.004) and 72% (p = 0.000001) respectively. No overt toxicity was noted. SK inhibition, resulting in reduced intracellular S1P, is an exciting novel anti-leukemic strategy potentially adding to the repertoire of non-chemotherapeutic agents for the treatment of ALL. Combinations of SK inhibitors with newer targeted agents show promise of greater leukemia reduction. Disclosures: No relevant conflicts of interest to declare.

Synergistic Activity of ABT-199 with Conventional Chemotherapy and Dinaciclib in B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2631-2631 ◽  
Author(s):  
Felix Seyfried ◽  
Salih Demir ◽  
Klaus-Michael Debatin ◽  
Lueder Hinrich Meyer
Keyword(s):  
Cell Death ◽  
B Cell ◽  
Cell Lines ◽  
Single Agent ◽  
Chemotherapeutic Agents ◽  
Remission Induction ◽  
Synergistic Activity ◽  
Cell Precursor ◽  
Protein Levels ◽  
Ic50 Values

Abstract Over the past decades, the use of optimized multi-agent chemotherapy protocols and improved risk stratification have led to superior patient outcome. However, avoiding therapy-related toxicity, decreasing the relapse rate of currently almost 20 % and improving the outcome of relapsed patients remain challenging problems. Aberrant activity of pathways involved in the regulation of survival and cell death contribute to development of leukemia and therapy failure. Anti-apoptotic BCL-2 family proteins are key regulators of apoptosis providing a promising target for novel directed therapies. The BH3 mimetic ABT-199 binds to BCL-2, counteracts its anti-death function and leads to apoptosis induction by direct release of pro-death BCL-2 family proteins such as BAX. BH3 mimetics are currently used pre-clinically and in first clinical trials. However the intrinsic or acquired resistance indicates the need for predictive markers and for effective combination treatment strategies. In this study, we addressed the activity of ABT-199 in B cell precursor (BCP) ALL. The effects of ABT-199 as single agent and in combination with chemotherapeutic drugs used in remission-induction therapy of pediatric ALL were analyzed using BCP-ALL cell lines (n=5) and a set of patient-derived primograft samples (n=9) established by transplantation of primary leukemia cells obtained from pediatric BCP-ALL patients at diagnosis onto immunodeficient NOD/SCID mice. Since ABT-199 does not bind to all antiapoptotic BCL-2 family members, sparing MCL-1, we included the cyclin-dependent kinase (CDK) inhibitor dinaciclib, which also exerts MCL-1 inhibitory effects. Cell viability was determined by flowcytometry according to fw/sc criteria and half maximal inhibitory concentrations (IC50) were estimated upon exposure to single agents and drug combinations. Combination indices (CI) indicating synergistic or additive effects were estimated according to Chou-Talalay. BCL-2 and MCL-1 protein levels were investigated by western blot analysis. Despite sensitivity in most of the BCP-ALL cell lines with IC50 values in the nanomolar range (mean IC50, 212 nM), one cell line, Nalm-6, showed ABT-199 resistance with an IC50 >10 µM. Interestingly, low protein levels of BCL-2 and increased MCL-1 expression were identified in this insensitive line, in contrast to high level BCL-2 and low MCL-1 expression in ABT-199 sensitive leukemias, indicating a potential marker to identify sensitive and resistant ALL. A pronounced synergism of ABT-199 in combination with chemotherapeutic agents was found with vincristine (CI 0.31 and 0.40, cell lines RS4;11 and Nalm-6) and even strong synergism with asparaginase and dexamethasone in ABT-199 monotherapy resistant Nalm-6 cells (CI 0.002, 0.14). Strikingly, the combination of the MCL-1 inhibitor dinaciclib with ABT-199 was highly effective and revealed a strong synergism for both compounds (CI 0.03). When we addressed the effects of ABT-199 on primograft ALL samples isolated from mice with full-blown leukemia, a pattern of high sensitivity (IC50 from 16 to 156 nM) similar to the cell lines was observed. In contrast, peripheral blood (PB) samples from healthy control donors were resistant to ex vivo exposure with ABT-199 (n=3, IC50 values > 1 µM). However, one primograft sample derived from a high risk patient showed ABT-199 insensitivity with an IC50 value of > 1 µM similar to healthy PBMCs. This primograft sample was also characterized by low BCL-2/high MCL-1 protein expression similar to Nalm-6. Most importantly, this resistant leukemia was also rendered ABT-199 sensitive by co-treatment with dinaciclib (CI 0.04). Taken together, we found efficacy of ABT-199 in the majority BCP-ALL cell lines and patient-derived primograft ALL samples and ABT-199 synergized with conventional chemotherapeutic agents. ABT-199 resistant leukemias characterized by low BCL-2/high MCL-1 expression were resensitized by addition of dinaciclib leading to strong synergism. These data indicate effective cell death sensitization by ABT-199 and potential strategies to overcome ABT-199 resistance in BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Anti-Leukemia Activity of the Selective BCL-2 Inhibitor ABT-199 in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By MCL-1/BCL-2 Expression Serving As Biomarker for Treatment Response

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1081-1081
Author(s):  
Felix Seyfried ◽  
Salih Demir ◽  
Rebecca Hörl ◽  
Stefan Köhrer ◽  
Annika Scheffold ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
High Ratio ◽  
Cell Precursor ◽  
Free Survival ◽  
Ic50 Values

Abstract Despite superior outcome and survival of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), relapse occurs in 10-20% and is associated with poor outcome, clearly indicating future challenges including reduction of relapse rates and effective treatment of reoccurred leukemia. Deficiencies in cell death and survival pathways have been implicated in therapy failure and treatment resistance in BCP-ALL. Members of the BCL-2 family are key regulators of these pathways and are therefore of interest as therapeutic targets. The small molecule ABT-199 binds selectively to BCL-2, inhibits its anti-apoptotic function and leads to release of pro-apoptotic molecules. Recently, ABT-199 has demonstrated clinical activity, particularly in poor prognosis CLL. However, insensitivity and resistance in different cases clearly emphasize the need of predictive markers for upfront identification of ABT-199 responsive leukemias. Here, we analyzed sensitivity for ABT-199 in a series of individual BCP-ALL samples, addressed mechanisms of resistance and evaluated markers indicating response to ABT-199. Anti-leukemic activities of ABT-199 were investigated in BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primograft samples (n=17), which were established by transplantation of primary patient ALL cells obtained at diagnosis onto NOD/SCID mice. Half maximal inhibitory concentrations (IC50) for ABT-199 were analyzed for each sample. Expression of apoptosis regulating molecules was investigated by western blot analysis and associated with ABT-199 responsiveness. Two MCL-1 deficient ALL cell lines were generated by CRISPR/Cas9 gene editing. Leukemia free-survival of ALL bearing animals was analyzed after in vivoABT-199 treatment. The majority of BCP-ALL samples showed sensitivity for ABT-199 induced cell death in the nanomolar range, both in cell lines (n=4, IC50: 29 - 422 nM) and patient-derived primograft samples (n=10, IC50: 1.7 - 74 nM), while 2 cell lines and 7 primograft leukemias showed insensitivity with IC50 values above 1 µM. ABT-199 binds directly to BCL-2 and upon binding, pro-apoptotic Bcl-2 family molecules like Bim are dislocated from BCL-2 and induce apoptosis. The anti-apoptotic BCL-2 family member MCL-1 is not bound by ABT-199, but sequesters pro-apoptotic molecules dislocated from BCL-2 leading to interruption of apoptosis induction. Therefore, we addressed expression levels of BCL-2 and MCL-1. We found high BCL-2 levels in ABT-199 sensitive and low BCL-2 levels in resistant leukemia samples and an opposite pattern for MCL-1 (high in resistant and low MCL-1 in sensitive ALL), in line with previous reports. Most interestingly, a high ratio of MCL-1 to BCL-2 expression (high MCL-1, low BCL-2) was significantly associated with high IC50 values/resistance (Spearman Rho correlation, p= .01), whereas a low MCL-1/BCL-2 ratio indicated ABT-199 sensitivity. Two of the 6 cell lines showed ABT-199 resistance (IC50 > 1 µM) and high Mcl-1 expression. Effective MCL-1 knock-out in both cell lines led to a clear sensitization for ABT-199 with up to 40-fold reduced IC50 values, clearly indicating MCL-1 as a key mediator of ABT-199 resistance in BCP-ALL. Finally, we also evaluated the anti-leukemia activity of ABT-199 in a preclinical setting in vivo. Two patient-derived leukemias, one with a low MCL-1/BCL-2 ratio of 0.9 and the other with a high ratio of 16.1, indicative of ABT-199 sensitivity or resistance, were transplanted onto NOD/SCID mice and treated with ABT-199 for 10 days after ALL engraftment. Most interestingly, a significantly increased leukemia free survival was observed in ABT-199 as compared to vehicle treated recipients (p<0.001) of the leukemia with the low MCL-1/BCL-2 ratio, in contrast to similar survival times of vehicle or ABT-199 treated animals bearing the high MCL-1/BCL-2 ratio ALL, clearly showing the predictive value of BCL-2 and MCL-1 levels in BCP-ALL. Taken together, ABT-199 shows anti-leukemia activity in the majority of BCP-ALL samples, with a strong association of high BCL-2 and low MCL-1 levels with ABT-199 sensitivity. Silencing of MCL-1 clearly revealed a crucial role for MCL-1 as mediator of ABT-199 resistance. Importantly, in vivo evaluation of ABT-199 in a preclinical setting highlighted the predictive value of BCL-2/MCL-1 expression for the identification of patients who would benefit from future BCL-2 directed therapies. Disclosures Stilgenbauer: Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding.

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