scholarly journals BRD4 Proteolysis Targeting Chimera (PROTAC) ARV-825 Targets Both NOTCH1-MYC Regulatory Circuit and Leukemia-Microenvironment in T-ALL

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 716-716
Author(s):  
Sujan Piya ◽  
Hong Mu ◽  
Seemana Bhattacharya ◽  
Teresa McQueen ◽  
Richard E Davis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
New Haven ◽  
Down Regulation ◽  
Regulatory Circuit ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Background: Salvage options for patients with relapsed T cell acute lymphoblastic leukemia (T-ALL) are limited, with less than 25% of these patients achieving second remission 1, 2. 70% of T-ALL cases have activating mutations of the NOTCH1 pathway, which transcriptionally activates MYC by binding to its `superenhancer' region 3, 4. Other deregulated oncogenic pathways in T-ALL include PI3K/Akt, the anti-apoptotic Bcl-2 family, and CDKN2A/2B cell cycle regulators 5, 6. The NOTCH1-MYC regulatory circuit is an attractive therapeutic target, but clinical development of gamma-secretase inhibitors (GSI) to target NOTCH1 has been limited by 'on target' toxicities. A better target may be BRD4, a critical component of superenhancer complexes that binds to acetylated histone (3 and 4) and drives NOTCH1 mediated MYC transcription7. ARV-825 is a hetero-bifunctional PROteolysis TArgeting Chimera (PROTAC) that has 3 components: a thienodiazepine-based BRD4 ligand, a linker arm, and a cereblon-binding ligand. ARV-825 recruits BRD4 to the E3 ubiquitin ligase cereblon and leads to efficient and sustained degradation of BRD4, resulting in down-regulation of MYC. Methods: We investigated the effectiveness of ARV-825 against T-ALL cell lines, including GSI-resistant lines. Since microenvironmental signals are critical for the survival of T-ALL, we specifically tested the impact of BRD4 degradation on CD44/CD44v, which integrates cell-extrinsic microenvironmental signals and is part of cysteine transporter that maintains low intra-cellular reactive oxygen species (ROS), necessary for T-ALL survival and the persistence of disease. We also examined the anti-leukemic effect of ARV-825 in a T-ALL patient-derived xenograft (PDX) mouse model of disseminated leukemia with a constitutively active NOTCH1 mutation. Results: The IC50s for all tested T-ALL cell lines at 72 hours were in the low nanomolar range (< 50 nM). ARV-825 leads to sustained degradation of BRD4 and down-regulation of its transcriptional targets MYC, Bcl-2 and Bcl-XL and inhibits cell proliferation and induces apoptosis in GSI-sensitive (HPB-ALL, KOPT1) and GSI-resistant (MOLT4, SUPT1) cell lines. Mass cytometry based proteomic analysis (CyTOF) and immunoblotting showed that ARV-825 down-regulated cell intrinsic oncogenic molecules: transcription factors Myc and NFkB, cell cycle regulator CDK6, activated PI3K/Akt, and anti-apoptotic Bcl2 family proteins. In addition ARV-825 down regulated two key molecules involved in leukemia-stroma interaction; CD44 (Fig. 1), and CD98, a component of amino acid transporters xCT, LAT1 and 2, both essential in regulation of oxidative stress. Quantitative PCR and immunoblotting analysis confirmed the transcriptional down regulation of total CD44 and CD44 variants 8-10 (2-fold change treated vs . untreated). As a functional correlate of down-regulation of CD98/CD44/CD44v, flow cytometry confirmed increased intracellular ROS generation (Fig. 2). Finally, in a PDX mouse model of human T-ALL, ARV-825 treatment resulted in lower leukemia burden (confirmed by flow cytometry for human CD45+ cells in bone marrow) and better survival compared to vehicle-treated control mice (p=0.002) (Fig.3). Reference: 1. Marks DI, Rowntree C. Management of adults with T-cell lymphoblastic leukemia. Blood 2017; 129(9): 1134-1142. 2. Litzow MR, Ferrando AA. How I treat T-cell acute lymphoblastic leukemia in adults. Blood 2015; 126(7): 833-41. 3. Sanchez-Martin M, Ferrando A. The NOTCH1-MYC highway toward T-cell acute lymphoblastic leukemia. Blood 2017; 129(9): 1124-1133. 4. Demarest RM, Ratti F, Capobianco AJ. It's T-ALL about Notch. Oncogene 2008; 27(38): 5082-91. 5. Girardi T, Vicente C, Cools J, De Keersmaecker K. The genetics and molecular biology of T-ALL. Blood 2017; 129(9): 1113-1123. 6. Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC et al. Notch signaling mediates G1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood 2009; 113(8): 1689-98. 7. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153(2): 320-34. Disclosures Qian: 4Arvinas, LLC. New Haven, CT: Employment. Raina: 4Arvinas, LLC. New Haven, CT: Employment. McKay: 6 ImmunoGen, Inc.Waltham, MA: Employment. Kantarjian: Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding. Andreeff: Daiichi Sankyo: Consultancy.

scholarly journals Therapeutic Stratification of PTEN-MYC Axis Appears to be Promising for Molecular Targeted Therapies in Refractory and Relapsed T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3883-3883
Author(s):  
Heinz Ahlert ◽  
Sanil Bhatia ◽  
Marc Remke ◽  
Melf Sönnichsen ◽  
Niklas Dienstbier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Drug Combinations ◽  
Killing Effect ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Lines

Therapy refractory and relapsed T-cell acute lymphoblastic leukemia (T-ALL) is still one of the most common causes for cancer related death in children. T-ALL is a highly heterogeneous disease with several established biomarkers, which promote its progression. These biomarkers are promising tools for patient stratification and can be used for targeted therapeutic approaches. For instance, PTEN is one of the most displayed mutations in pediatric T-ALL and plays an important role in proteolysis of the oncoprotein MYC. Therefore, it is not surprising that PTEN activity in T-ALL patients correlates with higher MYC protein levels. Importantly, PTEN mutations have recently been associated with poor overall survival in T-ALL patients, underscoring its importance for future treatment options using molecular targeted therapies. In this study, we validated the importance of PTEN as a MYC regulator in T-ALL and elucidated PTEN mutational status as a stratification marker for T-ALL patients. Comparative quantification of PTEN, phosphorylated AKT and MYC in leukemic T-cell lines (n=10) was analyzed at protein level, relative to healthy T-cells. PTEN loss or deceased expression was observed in half of the screened leukemic T-cell lines with concomitant high MYC expression and increased AKT phosphorylation. To identify the drug sensitivity of PTEN mutant (PTENm) and PTEN wild type (PTENwt) cell lines, high-throughput drug screening was performed with 180 inhibitors, including among others kinase-, BET- and gamma secretase inhibitors, as well as conventional chemotherapeutics. PTENm cell lines showed remarkable sensitivity in nanomolar ranges against PI3K inhibitors compared to PTENwt (P=0.001). Anticipating that tumor cells eventually escape the killing effect of single pathway targeted drugs, the need for molecular identification and characterization of synergistic drug combinations is evident. Therefore, diverse compound combinations were tested in an 11 by 11 matrix of different concentrations in PTENm and PTENwt cell lines. Synergism was predicted with Combenefit software using Loewe model to assess putative additive versus synergistic effects. We observed that simultaneous use of copanlisib (PI3K inhibitor) with JQ1 (BET inhibitor) had a strong killing effect in PTENm cell lines. To assess target downregulation after 24 hours of drug treatment, lysates were taken and visualized using western blot. A strong decrease of MYC expression was observed already with a combination of low concentrations of copanlisib and JQ1 with subsequent higher apoptosis induction as measured by Annexin V/PI staining. A second drug combination that showed synergy in PTENm was observed with copanlisib and alisertib, an Aurora kinase-A (AURKA) inhibitor, expecting changes in cell cycle and reduced MYC stability. Indeed, results of cell cycle analysis have revealed an inhibition during mitosis and cellular MYC levels were decreased, suggesting that this combination affects highly proliferative MYC-dependent tumor cells. Contrary to PTENm, in PTENwt cell lines, PI3K inhibition alone showed no cytotoxic effect, but synergy was detected in combination with the MEK inhibitor cobimetinib. Moreover, in this synergistic combination MYC was effectively downregulated on protein level accompanied with changes in regulatory pathways including AKT and MEK. Interestingly, phosphorylation of MYC changed from the stabilizing site (S62) to the degradative site (T58), proposing the progress of MYC degradation. In summary, we observed the importance of PTEN as a regulator for MYC proteolysis and related pathways including AKT and MEK. The loss of PTEN is accompanied with increased MYC. Synergy studies have observed effective drug combinations for PTENm and PTENwt with the aim to downregulate MYC and induce apoptosis in tumor cells. Based on these findings, PTEN-MYC axis seems suitable as a potential stratification marker for future therapy options in refractory and relapsed T-ALL. In the next step, promising drug combinations will be tested in a patient derived xenograft mouse model to validate these findings. Figure Disclosures No relevant conflicts of interest to declare.

Frequency of NUP214-ABL1 Oncogene in Patients with T-Cell Acute Lymphoblastic Leukemia (T-ALL) and Analysis of the Activity of Imatinib and Nilotinib in NUP214-ABL1-Expressing T-ALL Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 710-710
Author(s):  
Alfonso Quintas-Cardama ◽  
Weigang Tong ◽  
Taghi Manshouri ◽  
Jan Cools ◽  
D. Gary Gilliland ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Inhibitory Activity ◽  
Lymphoblastic Leukemia ◽  
Fusion Transcript ◽  
Lymphoblastic Lymphoma ◽  
Abl Kinase ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Kinase Inhibitory Activity

Abstract The fusion of ABL1 with BCR results in the hybrid BCR-ABL1 oncogene that encodes the constitutively active Bcr-Abl tyrosine kinase encountered in the majority of patients with chronic myeloid leukemia (CML) and in approximately 30% of pts with B-cell acute lymphoblastic leukemia (B-ALL). Recently, the episomal amplification of ABL1 has been described in 6% of pts with T-ALL (Nat Genet2004;36:1084–9). Molecular analysis demonstrated the oncogenic fusion of ABL1 with the nuclear pore complex protein NUP214 (NUP214-ABL1). We screened 29 pts with T-cell lymphoblastic lymphoma (T-LBL) and T-ALL for the presence of the NUP214-ABL1 fusion transcript by RT-PCR using specific primers for the 5 different transcripts thus far described. Three (10%) pts were found to express this fusion transcript, including 2 with T lymphoblastic lymphoma (NUP214 exon 31) and 1 with T-ALL (NUP214 exon 29). This was confirmed by direct sequencing in all cases. All pts received therapy with hyperCVAD and achieved a complete remission (CR). However, 2 of them died 6 and 9 months into therapy, respectively. One other pt remains in CR (19+ months) by morphologic and flow cytometry criteria. However, NUP214-ABL1 is still detectable in peripheral blood by nested PCR, thus suggesting minimal residual disease (MRD). We then studied the activity of the tyrosine kinase inhibitors imatinib and nilotinib in the NUP214-ABL1-expressing cell lines PEER and BE-13. Although PEER and BE-13 cell viability was reduced with both agents, the IC50 was almost 10-fold higher for imatinib (643 nM) than for nilotinib (68 nM) (F test, p<0.001), which parallels the 10− to 30− fold higher Abl kinase inhibitory activity of nilotinib compared to imatinib in BCR-ABL-expressing cells. Nilotinib also potently inhibited the cell proliferation of BE-13 cells (IC50 131 nM). In contrast, Jurkat cells, a T-ALL cell line which does not carry NUP214-ABL1, were remarkably resistant to both imatinib and nilotinib with an IC50 values greater than 5 μM indicating that the cytotoxicity mediated by both TKIs is not related to a general toxic effect on T-ALL cell lines. The inhibition of cellular proliferation by imatinib and nilotinib was associated with a dose- and time-dependent induction of apoptosis in both PEER and BE-13 cells. In Western blotting, higher inhibition of phospho-Abl and phospho-CRKL (a surrogate of Bcr-Abl kinase status) was observed in PEER cells upon exposure to nilotinib as compared with imatinib at their respective IC50 concentrations for cell growth inhibition. We conclude that NUP214-ABL1 can be detected in 10% of pts with T-cell malignancies and its detection can be used as a sensitive marker of MRD. Imatinib and nilotinib potently inhibits the growth of NUP214-ABL1-expressing cells. Given the higher Abl kinase inhibitory activity of nilotinib with respect to imatinib, this agent must be further investigated in clinical studies targeting patients with T-ALL and T-LBL expressing the NUP214-ABL1 fusion kinase.

Activation of the JNK Pathway Mediates Chemoresistance in T-Cell Acute Lymphoblastic Leukemia by Phosphorylation and Proteosomal Degradation of BimEL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2372-2372
Author(s):  
Kam Tong Leung ◽  
Karen Kwai Har Li ◽  
Samuel Sai Ming Sun ◽  
Paul Kay Sheung Chan ◽  
Yum Shing Wong ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cytochrome C ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Chemotherapeutic Agents ◽  
Jnk Pathway ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Induced Apoptosis

Abstract Despite progress in the development of effective treatments against T-cell acute lymphoblastic leukemia (T-ALL), about 20% of patients still exhibit poor response to the current chemotherapeutic regimens and the cause of treatment failure in these patients remains largely unknown. In this study, we aimed at finding mechanisms that drive T-ALL cells resistant to chemotherapeutic agents. By screening etoposide sensitivity of a panel of T-ALL cell lines using DNA content and PARP cleavage as apoptosis markers, we identified an apoptosis-resistant cell line, Sup-T1. Western blot analysis and caspase activity assay showed that Sup-T1 cells were deficient in etoposide-induced activation of caspase-3 and caspase-9. In addition, mitochondrial cytochrome c release was not evident in etoposide-treated Sup-T1 cells. However, addition of exogenous cytochrome c in cell-free apoptosis reactions induced prominent caspase-3 activation, indicating that the chemoresistance observed in Sup-T1 cells was due to its insusceptibility to the drug-induced mitochondrial alterations. Analysis of the basal expression of the Bcl-2 family proteins revealed that the levels of Bcl-2 was higher in Sup-T1 cells, while Bax and BimEL levels were lower, when compared to etoposide-sensitive T-ALL cell lines. Gene silencing using antisense oligonucleotide to Bcl-2 and overexpression of Bax did not resensitize cells to etoposide-induced apoptosis. On the contrary, transient transfection of BimEL into Sup-T1 cells significantly restored etoposide sensitivity. Further experiments revealed that the lack of BimEL expression in Sup-T1 cells was due to the rapid degradation of newly-synthesized BimEL by the proteosomal pathway, as treatment of Sup-T1 cells with a proteosome inhibitor significantly restored the protein level of BimEL. Moreover, treatment with proteosome inhibitor resulted in mobility shift of BimEL, which was sensitive to phosphatase digestion. Furthermore, treatment of Sup-T1 cells with JNK inhibitor resulted in accumulation of BimEL, and pretreatment with JNK inhibitor restored sensitivity of Sup-T1 cells to etoposide-induced apoptosis, indicating that constitutive activation of the JNK pathway in Sup-T1 cells was responsible for promoting BimEL phosphorylation, and this may serve as a signal targeting BimEL to the proteosome for degradation. Altogether, our findings provide the first evidence that JNK activation correlates inversely with BimEL level by promoting its phosphorylation and degradation. This, in turn, reduces the sensitivity of T-ALL cells to chemotherapeutic agents.

Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4630-4630
Author(s):  
Samuel D Gusscott ◽  
Florian Kuchenbauer ◽  
Andrew P Weng
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Notch Signaling ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Gamma Secretase ◽  
Protein Levels ◽  
Cell Acute Lymphoblastic Leukemia ◽  
T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

Down-Regulation of Mir-26b and Its Inhibition on the Growth of T Cell Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2440-2440
Author(s):  
Tian Yuan ◽  
Yaling Yang ◽  
Jeffrey You ◽  
Daniel Lin ◽  
Kefeng Lin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Growth ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Level ◽  
Rt Pcr ◽  
Altered Expression ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Introduction: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy accounting for 15% of pediatric and 25% of adult acute lymphoblastic leukemia (ALL) cases. With current chemotherapies and transplantation therapy, there are still 25-50% T-ALL patients that suffer from relapse and have a poor outcome. MicroRNAs (miRNAs or miRs) are endogenous small non-coding RNAs (containing about 22 nucleotides in length). miRs function at posttranscriptional level as negative regulators of gene expression and exert their regulatory function through binding to target mRNAs and silencing gene expression. To better understand the pathogenesis and develop the new therapeutic targets of T-ALL, we have developed a Pten tumor suppressor knockout T-ALL mouse model and profiled miRs from the mouse Pten deficient T-ALL. miR-26b was one of the miRs that were found down-regulated in the mouse Pten deficient T-ALL. Recent studies showed that the aberrant expression of miR-26b is implicated in several types of cancer. The expression level of miR-26b and its role of in T-ALL, however, are unknown. We investigated if the expression level of miR-26b is aberrant in T-ALL and the effect of potentially altered expression on the growth of human T-ALL cells. Methods: We conducted miR array profiling to identify differentially expressed miRs in the mouse Pten deficient T-ALLs compared with preneoplastic thymocyte controls. We validated expression levels of several miRs, including miR-26b, that are differentially expressed in mouse and human T-ALL cells using quantitative RT-PCR. We also overexpressed miR-26b using a lentivirus based vector in human T-ALL cell lines to assess its effect on cell growth and apoptosis. Results: Employing miR array profiling, we identified a subset of miRs that exhibited marked altered expression in the mouse Pten deficient T-ALL cells. Quantitative RT-PCR validated that the expression level of miR-26b in the mouse Pten deficient T-ALL cells was markedly lower in comparison to that of preneoplastic thymocytes. To determine if miR-26b expression level is also altered in human T-ALL, we performed quantitative RT-PCR on a panel of human T-ALL cell lines. Indeed, the expression level of miR-26b is significantly lower in the human T-ALL cell lines when compared with that of normal thymocytes. To functionally assess if miR-26b plays a role in the cell growth of human T-ALL cells, we expressed exogenous miR-26b in a panel of human T-ALL cell lines. We demonstrated that the expression of exogenous miR-26b significantly reduced the proliferation and promoted apoptosis of several human T-ALL cell lines. Conclusions: Our results demonstrated that miR-26b is down-regulated in T-ALL and the expression of exogenous miR-26b elicits deceased cell proliferation and increased apoptosis of human T-ALL. These results suggest that miR-26b may function as a tumor suppressor in the development of T-ALL and further characterization of the target and regulation of miR-26b may have therapeutic implications. Disclosures No relevant conflicts of interest to declare.

scholarly journals Exploring the ATR-CHK1 pathway in the response of doxorubicin-induced DNA damages in acute lymphoblastic leukemia cells

2021 ◽  
Author(s):  
Andrea Ghelli Luserna Di Rorà ◽  
Martina Ghetti ◽  
Lorenzo Ledda ◽  
Anna Ferrari ◽  
Matteo Bocconcelli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Cycle Checkpoint ◽  
M Cell ◽  
Dna Damages ◽  
Cycle Checkpoint ◽  
Dna Double Helix

AbstractDoxorubicin (Dox) is one of the most commonly used anthracyclines for the treatment of solid and hematological tumors such as B−/T cell acute lymphoblastic leukemia (ALL). Dox compromises topoisomerase II enzyme functionality, thus inducing structural damages during DNA replication and causes direct damages intercalating into DNA double helix. Eukaryotic cells respond to DNA damages by activating the ATM-CHK2 and/or ATR-CHK1 pathway, whose function is to regulate cell cycle progression, to promote damage repair, and to control apoptosis. We evaluated the efficacy of a new drug schedule combining Dox and specific ATR (VE-821) or CHK1 (prexasertib, PX) inhibitors in the treatment of human B−/T cell precursor ALL cell lines and primary ALL leukemic cells. We found that ALL cell lines respond to Dox activating the G2/M cell cycle checkpoint. Exposure of Dox-pretreated ALL cell lines to VE-821 or PX enhanced Dox cytotoxic effect. This phenomenon was associated with the abrogation of the G2/M cell cycle checkpoint with changes in the expression pCDK1 and cyclin B1, and cell entry in mitosis, followed by the induction of apoptosis. Indeed, the inhibition of the G2/M checkpoint led to a significant increment of normal and aberrant mitotic cells, including those showing tripolar spindles, metaphases with lagging chromosomes, and massive chromosomes fragmentation. In conclusion, we found that the ATR-CHK1 pathway is involved in the response to Dox-induced DNA damages and we demonstrated that our new in vitro drug schedule that combines Dox followed by ATR/CHK1 inhibitors can increase Dox cytotoxicity against ALL cells, while using lower drug doses. Graphical abstract • Doxorubicin activates the G2/M cell cycle checkpoint in acute lymphoblastic leukemia (ALL) cells. • ALL cells respond to doxorubicin-induced DNA damages by activating the ATR-CHK1 pathway. • The inhibition of the ATR-CHK1 pathway synergizes with doxorubicin in the induction of cytotoxicity in ALL cells. • The inhibition of ATR-CHK1 pathway induces aberrant chromosome segregation and mitotic spindle defects in doxorubicin-pretreated ALL cells.

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