Lymphoblastic Leukemia Cell Line Research Articles

Deep thermal profiling for detection of functional proteoform groups.

The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.

FBXW7 tumor suppressor regulation by dualspecificity tyrosine-regulated kinase 2

FBXW7 is a member of the F-box protein family, which functions as the substrate recognition component of the SCF E3 ubiquitin ligase. FBXW7 is a main tumor suppressor due to its ability to control proteasome-mediated degradation of several oncoproteins such as c-Jun, c-Myc, Cyclin E1, mTOR, and Notch1-IC. FBXW7 inactivation in human cancers results from a somatic mutation or downregulation of its protein levels. This work describes a novel regulatory mechanism for FBXW7 dependent on the serine/threonine protein kinase DYRK2. We show that DYRK2 interacts with and phosphorylates FBXW7 resulting in its proteasome-mediated degradation. DYRK2-dependent FBXW7 destabilization is independent of its ubiquitin ligase activity. The functional analysis demonstrates the existence of DYRK2-dependent regulatory mechanisms for key FBXW7 substrates. Finally, we provide evidence indicating that DYRK2-dependent regulation of FBXW7 protein accumulation contributes to cytotoxic effects in response to chemotherapy agents such as Doxorubicin or Paclitaxel in colorectal cancer cell lines and to BET inhibitors in T-cell acute lymphoblastic leukemia cell lines. Altogether, this work reveals a new regulatory axis, DYRK2/FBXW7, which provides an understanding of the role of these two proteins in tumor progression and DNA damage responses.

Evaluation the Effect of Chemotherapy Drugs and Thiosemicarbazone Complexes on the Expression of URHC and CASC15 LncRNAs in Acute Lymphoblastic Leukemia Cell Line

Evaluation the Effect of Chemotherapy Drugs and Thiosemicarbazone Complexes on the Expression of URHC and CASC15 LncRNAs in Acute Lymphoblastic Leukemia Cell Line

Use of Novel Triphenyltin (IV) Dithiocarbamate Compounds in Assessing the Cytotoxicity and Mode of Cell Death of Acute Lymphoblastic Leukemia Cell Lines, CCRF CEM (CCL-119)

Chemotherapy and radiotherapy are often used to treat Acute Lymphoblastic Leukemia (ALL). However, the resistance problem of treatment using the current drug in ALL patients has decreased the effectiveness of such therapy. Therefore, a study needs to be conducted to identify the potential of new anticancer agents. Organotin (IV) dithiocarbamate, an organometallic compound, causes toxic effects on cancer cells in vitro and in vivo. This study examines the cytotoxic effects and mode of cell death of novel organotin (IV) dithiocarbamate compounds of triphenyltin (IV) diisopropyldithiocarbamate (compound 1), triphenyltin (IV) diallyldithiocarbamate (compound 2) and triphenyltin (IV) diethyldithiocarbamate (compound 3) on the acute lymphoblastic leukemia cell line, CCRF CEM (CCL-119). An MTT assay was used for the determination of the cytotoxicity of the compounds by treating cells with compounds 1-3 at the highest concentration of 10 µm for 24 h. The induction of the mode of cell death for compounds 1, 2, and 3 were identified using Annexin V-FITC/PI assay. From the results, all triphenyltin (IV) dithiocarbamate compounds have substantial cytotoxic effects with IC50 values between 0.18-0.20 μm. The chemicals induced apoptosis cell death in CCL-119 cells, based on the determination of cell death mode. However, statistical analysis showed that only apoptotic cell death for treatment with compounds 1-2 is significant compared to negative controls. In conclusion, all these compounds have a high cytotoxicity effect and are able to cause cell death via apoptosis in CCL-119 cells.

LncRNA15691 promotes T-ALL infiltration by upregulating CCR9 via increased MATR3 stability.

Our previous studies demonstrated that CCR9 plays an important role in several aspects of T-cell acute lymphoblastic leukemia progression and that CCR9 is a potential therapeutic target. However, the underlying mechanism that regulates CCR9 expression remains incompletely understood. In this study, bioinformatics analysis and validation in clinical samples revealed the lncRNA15691 to be positively correlated with CCR9 mRNA expression and significantly upregulated in T-cell acute lymphoblastic leukemia samples and CCR9high T-cell acute lymphoblastic leukemia cell lines. LncRNA15691, a previously uncharacterized lncRNA, was found to be located in both the cytoplasm and the nucleus via fluorescence in situ hybridization assay. In addition, lncRNA15691 upregulated the expression of CCR9 and was involved in T-cell acute lymphoblastic leukemia cell invasion. In vivo experiments showed that lncRNA15691 promoted leukemia cell homing/infiltration into the bone marrow, blood, and spleen, whereas the CCR9 ligand, CCL25, augmented the extramedullary infiltration of CCR9low leukemia cells overexpressing lncRNA15691 into blood, spleen, and liver. Subsequently, RNA protein pull-down assays, coupled with liquid chromatography-tandem mass spectrometry, were used to uncover potential lncRNA15691-interacting proteins, which were then validated by RNA immunoprecipitation. These mechanistic studies revealed that lncRNA15691 upregulated CCR9 expression via directly binding to and stabilizing MATR3 by inhibiting its nuclear degradation mediated by PKA. Collectively, our study revealed a novel mechanism of regulating CCR9 expression and implicated lncRNA15691 as a potential novel biomarker for T-cell acute lymphoblastic leukemia infiltration.

CD4 CAR-T cells targeting CD19 play a key role in exacerbating cytokine release syndrome, while maintaining long-term responses

BackgroundTo date, T cells redirected with CD19-specific chimeric antigen receptors (CAR) have gained impressive success in B-cell malignancies. However, treatment failures are common and the occurrence of severe toxicities, such...

MiR-29a-3p Targets Hepatoma-Derived Growth Factor to Inhibit the Proliferation and Promote the Apoptosis of E6-1 Cells

To investigate the regulatory effect of miRNA-29a-3p (miR-29a-3p ) on hepatoma-derived growth factor (HDGF), and its influences on the proliferation and apoptosis of acute lymphoblastic leukemia (ALL) cell line E6-1. Thirty-five patients with ALL treated in our hospital from January 2017 to January 2019 were selected as research objects, and 35 adults who underwent physical examination in the same period were selected as healthy control group. The miR-29a-3p overexpression vector, miR-29a-3p inhibitory expression vector, and miR-29a-3p and HDGF co-overexpression vector were transfected into E6-1 cells. The expression levels of miR-29a-3p and HDGF mRNA were detected by RT-qPCR. The expression of protein was detected by Western blot. The targeting relationship between miR-29a-3p and HDGF was detected by dual luciferase reporter assay. The cell proliferation was detected by CCK-8 method, while apoptosis detected by flow cytometry. Compared with healthy control group, HDGF was highly expressed in serum of patients with ALL and leukemia cells HuT 78, E6-1, CCRF-CEM, while miR-29a-3p was low expressed (P<0.05). After overexpression of miR-29a-3p , the expression levels of CyclinD1 and Bcl-2 in leukemia cells E6-1 were significantly reduced, while the expression levels of p21 and Bax were significantly increased (P<0.05). The activity of E6-1 cells was also significantly reduced, while the apoptosis rate of E6-1 cells was significantly increased (P<0.05). miR-29a-3p could target and regulate the expression of HDGF, while overexpression of HDGF reversed the inhibitory effect of miR-29a-3p overexpression on the proliferation and promotion effect on the apoptosis of leukemia cells E6-1. Overexpression of miR-29a-3p can inhibit the proliferation and promote the apoptosis of ALL cells E6-1, and its mechanism may be related to the regulation of HDGF expression.

Glutathione levels are associated with methotrexate resistance in acute lymphoblastic leukemia cell lines.

Methotrexate (MTX), a folic acid antagonist and nucleotide synthesis inhibitor, is a cornerstone drug used against acute lymphoblastic leukemia (ALL), but its mechanism of action and resistance continues to be unraveled even after decades of clinical use. To better understand the mechanisms of this drug, we accessed the intracellular metabolic content of 13 ALL cell lines treated with MTX by 1H-NMR, and correlated metabolome data with cell proliferation and gene expression. Further, we validated these findings by inhibiting the cellular antioxidant system of the cells in vitro and in vivo in the presence of MTX. MTX altered the concentration of 31 out of 70 metabolites analyzed, suggesting inhibition of the glycine cleavage system, the pentose phosphate pathway, purine and pyrimidine synthesis, phospholipid metabolism, and bile acid uptake. We found that glutathione (GSH) levels were associated with MTX resistance in both treated and untreated cells, suggesting a new constitutive metabolic-based mechanism of resistance to the drug. Gene expression analyses showed that eight genes involved in GSH metabolism were correlated to GSH concentrations, 2 of which (gamma-glutamyltransferase 1 [GGT1] and thioredoxin reductase 3 [TXNRD3]) were also correlated to MTX resistance. Gene set enrichment analysis (GSEA) confirmed the association between GSH metabolism and MTX resistance. Pharmacological inhibition or stimulation of the main antioxidant systems of the cell, GSH and thioredoxin, confirmed their importance in MTX resistance. Arsenic trioxide (ATO), a thioredoxin inhibitor used against acute promyelocytic leukemia, potentiated MTX cytotoxicity in vitro in some of the ALL cell lines tested. Likewise, the ATO+MTX combination decreased tumor burden and extended the survival of NOD scid gamma (NSG) mice transplanted with patient-derived ALL xenograft, but only in one of four ALLs tested. Altogether, our results show that the cellular antioxidant defense systems contribute to leukemia resistance to MTX, and targeting these pathways, especially the thioredoxin antioxidant system, may be a promising strategy for resensitizing ALL to MTX.

Combined Inhibition of MCL1 By AZD5991 and BCL2/Bclxl By AZD4320 As Promising Strategies to Overcome Acute Leukemia Tumor Burden and Niche Chemoresistance

Background. Abnormal regulation of anti-apoptotic proteins belonging to the BCL2 family contributes to therapeutic resistance of many hematological neoplasms. Although Venetoclax, a selective inhibitor of the BCL2 protein, has proven to exert beneficial effects in acute leukemias, the activation of another anti-apoptotic protein, MCL1, a key factor to Venetoclax acquired resistance, frequently reduces its benefits. Additionally, it is known that BCL-xL plays an important pro-survival role in cancer cells by interacting with BCL-2 Hence, we hypothesize that the co-inhibition of MCL1, Bcl-2 and BCL-xL would represent an improved strategy for the treatment of patients with acute leukemia. Thus, in this study, we assessed the efficacy of specific inhibitors of MCL1, BCL2 and BCLxL antiapoptotic proteins. Aims. To investigate the in vitro, ex vivo and in vivo effects of AZD5991 (AstraZeneca), a novel MCL1 inhibitor compound, alone or in combination with AZD4320 (BCL-2/BCL-XL inhibitor compound), venetoclax, cytarabine and doxorubicin. Methods.MCL1 mRNA expression data were obtained from TCGA AML study, performed in mononuclear cells isolated from 163 AML patients (median age 57.5 years [range: 18-88]). Bone marrow (BM) aspirates (25) from healthy donors (HD), Myelodysplastic syndrome (MDS), de novo AML, and acute lymphoblastic leukemia (ALL) patients at diagnosis were submitted to analysis of MCL1 protein levels by flow cytometry and immunofluorescence (IF). A panel of myeloid (OCI-AML3, MOLM13, U937, HL60, KG1a, K562) and lymphoblastic (RS4;11, Jurkat) leukemia cell lines were treated with increasing concentrations of AZD5991 for 48h and the 50% inhibiting concentration and apoptosis (annexin-V stain) rates were determined. Mononuclear and hematopoietic stem (CD34+) cells from HD were submitted to cytotoxic assays. Leukemia cell lines were also tested for reactive oxygen species (ROS) and nitric oxide (NO) production (DAF dye), mitochondrial membrane potential (DilC1 probe) and protein expression (flow cytometry, Western blot) in a time course of 0 to 48h, alone or in combination. Moreover, we tested the AZD5991/AZD4320 action in malignant cells cultured in a 3D-system (in the presence of the mesenchymal cells) and in a monocyte monolayer (2D) in order to evaluate tumor microenvironment (TME) participation. We also developed a xenograft mouse model using U937 leukemia cell line. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. The TCGA study (CBIO Portal) analysis showed high MCL1 gene expression in AML patients was observed (p<.0001). Immunofluorescence and flow cytometry showed that MCL1, BCL-2 and BCL-xL protein levels higher in MDS, AML and ALL BM cells compared to normal cells. All leukemia cell lines were dose-dependent sensitive to AZD5991. Growth inhibiting concentrations were OCI-AML3/0.04μM, U937/4.3μM, RS4;11/3.5μM, Jurkat/0.3μM. Apoptosis was increased in a time and concentration-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to impair mitochondrial membrane potential with reduced mitochondrial ROS and NO production (p<.01) and decreased MCL1, Bax and Bak but increased Bim protein levels. Coculture in a 3D-system showed that AZD5991 treatment reduced patients’ cell growth, without modulating HD cells. In the 2D-coculture, programmed monocytes intensified apoptosis with synergistic effect between AZD5991 and AZD4320. In vitro experiments demonstrated an additive effect between AZD5991 and AZD4320, venetoclax, cytarabine or doxorubicin. In addition, xenotransplanted mice confirmed the efficacy of AZD5991 compound alone or in combination with AZD4320 due to reduction of tumor weight (fold decrease (FD): 3.8; 2.8) and tumor volume (FD: 6.5; 2.8) compared to vehicle-treated group, respectively. Conclusion. MCL1 overexpression in mononuclear cells from patients with acute leukemia and that present or not resistance to venetoclax treatment, is associated with poor prognosis and relapse disease. Our results demonstrated that co-targeting MCL-1, BCL-2 and BCL-xL, through the AZD5991 and AZD4320 compounds in the treatment of acute leukemias, may enhance therapeutic specificity, overcome chemoresistance and contribute with the cure of these aggressive malignant disorders. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.

Comparative Genomic Hybridization and Transcriptome Sequencing Reveal Genes with Gain in Acute Lymphoblastic Leukemia: JUP Expression Emerges as a Survival-Related Gene.

Acute lymphoblastic leukemia (ALL) in children or adults is characterized by structural and numeric aberrations in chromosomes; these anomalies strongly correlate with prognosis and clinical outcome. Therefore, this work aimed to identify the genes present in chromosomal gain regions found more frequently in patients with acute lymphoblastic leukemia (ALL) and ALL-derived cell lines using comparative genomic hybridization (CGH). In addition, validation of the genes found in these regions was performed utilizing RNAseq from JURKAT, CEM, and SUP-B15 cell lines, as well as expression microarrays derived from a MILE study. Chromosomes with common gain zones that were maintained in six or more samples were 14, 17, and 22, in which a total of 22 genes were identified. From them, NT5C3B, CNP, ACLY, and GNB1L maintained overexpression at the mRNA level in the cell lines and in patients with ALL. It is noteworthy that SALL2 showed very high expression in T-ALL, while JUP was highly expressed in B-ALL lineages. Interestingly, the latter correlated with worse survival in patients. This provided evidence that the measurement of these genes has high potential for clinical utility; however, their expressions should first be evaluated with a sensitive test in a more significant number of patients.

Resistance to the BCL-XL degrader DT2216 in T-cell acute lymphoblastic leukemia is rare and correlates with decreased BCL-XL proteolysis.

The BCL-2 family of anti-apoptotic proteins, BCL-2, BCL-XL and MCL-1, can mediate survival of some types of cancer. DT2216 is a PROteolysis-TArgeting Chimera (PROTAC) that degrades BCL-XL specifically and is in phase 1 trials. We sought to define the frequency and mechanism of resistance to DT2216 in T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We measured cell survival and protein levels of BCL-XL, BCL-2, MCL-1 and the pro-apoptotic BIM in 13 distinct T-ALL cell lines after exposure to varying concentrations of DT2216. We identified concentrations of DT2216 which were cytotoxic to each T-ALL cell line. These concentrations have no correlation with the initial protein levels of BCL-XL, BCL-2, MCL-1 or BIM in each cell line. However, there was a correlation between survival to DT2216 and the efficiency of degradation of BCL-XL by DT2216. Only one cell line, SUP-T1, had significant resistance to DT2216, defined as an IC50 above what is achievable in murine tumors in vivo. Resistance to DT2216 is rare in a wide variety of T-ALL cells but when it occurs is correlated with decreased BCL-XL degradation. Resistance to DT2216 in T-ALL is not predicted by initial BCL-XL or BIM protein levels, or BCL-2 or MCL-1 levels before or after treatment. These data imply that a phase 2 clinical trial of DT2216 in T-ALL should be widely available and not limited to a subset of patients.

An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia

The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.

Acute lymphoblastic leukemia necessitates GSH-dependent ferroptosis defenses to overcome FSP1-epigenetic silencing

Ferroptosis is a form of cell death triggered by phospholipid hydroperoxides (PLOOH) generated from the iron-dependent oxidation of polyunsaturated fatty acids (PUFAs). To prevent ferroptosis, cells rely on the antioxidant glutathione (GSH), which serves as cofactor of the glutathione peroxidase 4 (GPX4) for the neutralization of PLOOHs. Some cancer cells can also limit ferroptosis through a GSH-independent axis, centered mainly on the ferroptosis suppressor protein 1 (FSP1). The significance of these two anti-ferroptosis pathways is still poorly understood in cancers from hematopoietic origin. Here, we report that blood-derived cancer cells are selectively sensitive to compounds that block the GSH-dependent anti-ferroptosis axis. In T- and B- acute lymphoblastic leukemia (ALL) cell lines and patient biopsies, the promoter of the gene coding for FSP1 is hypermethylated, silencing the expression of FSP1 and creating a selective dependency on GSH-centered anti-ferroptosis defenses. In-trans expression of FSP1 increases the resistance of leukemic cells to compounds targeting the GSH-dependent anti-ferroptosis pathway. FSP1 over-expression also favors ALL-tumor growth in an in vivo chick chorioallantoic membrane (CAM) model. Hence, our results reveal a metabolic vulnerability of ALL that might be of therapeutic interest.

Phospho-Specific Flow Cytometry Reveals Signaling Heterogeneity in T-Cell Acute Lymphoblastic Leukemia Cell Lines

Several signaling pathways are aberrantly activated in T-ALL due to genetic alterations of their components and in response to external microenvironmental cues. To functionally characterize elements of the signaling network in T-ALL, here we analyzed ten signaling proteins that are frequently altered in T-ALL -namely Akt, Erk1/2, JNK, Lck, NF-κB p65, p38, STAT3, STAT5, ZAP70, Rb- in Jurkat, CEM and MOLT4 cell lines, using phospho-specific flow cytometry. Phosphorylation statuses of signaling proteins were measured in the basal condition or under modulation with H2O2, PMA, CXCL12 or IL7. Signaling profiles are characterized by a high variability across the analyzed T-ALL cell lines. Hierarchical clustering analysis documents that higher intrinsic phosphorylation of Erk1/2, Lck, ZAP70, and Akt, together with ZAP70 phosphorylation induced by H2O2, identifies Jurkat cells. In contrast, CEM are characterized by higher intrinsic phosphorylation of JNK and Rb and higher responsiveness of Akt to external stimuli. MOLT4 cells are characterized by higher basal STAT3 phosphorylation. These data document that phospho-specific flow cytometry reveals a high variability in intrinsic as well as modulated signaling networks across different T-ALL cell lines. Characterizing signaling network profiles across individual leukemia could provide the basis to identify molecular targets for personalized T-ALL therapy.

Integrating Activity-Guided Strategy and Fingerprint Analysis to Target Potent Cytotoxic Brefeldin A from a Fungal Library of the Medicinal Mangrove Acanthus ilicifolius.

Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove Acanthus ilicifolius collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera Verticillium, Neocosmospora, Valsa, and Pyrenochaeta were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 μg/mL. Furthermore, the fungus Penicillium sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC50 values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds.

Abstract 3775: Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity

Abstract Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Recent studies have shown that some forms of hematologic malignancies (Ashton et al., 2018) and solid tumors (Molina et al., 2018) have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation. Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins leading to selective lethality in hematologic malignancies reliant on oxidative phosphorylation (Norberg et al., 2017). Combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant chronic myelogenous leukemia (CML), both in vitro and in vivo (Kuntz et al., 2017). We have previously reported that elatol, the major compound from algae Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary CML cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells treated with single-digit micromolar concentrations of elatol for short periods revealed strong downregulation of mitochondrion-encoded proteins (no transcriptional effect). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 2-10x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines. Through sedimentation property analysis using sucrose gradients, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation. Moreover, elatol treatment leads to activation of the integrated stress response (ISR) evidenced by induction of ATF4 and CHOP. We found that elatol activates the ISR through mitochondrial stress in which cleaved DELE1 binds to HRI to phosphorylate eIF2α, a mechanism described in Fessler et al., 2020. In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis and mechanistically different from chloramphenicol. Tigecycline’s compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol’s greatly improved potency provides a potential starting point for further optimization of this paradigm. Citation Format: Tyler A. Cunningham, Priyanka Maiti, Antonio Barrientos, Jonathan H. Schatz. Inhibition of mitochondrial translation by the marine natural product elatol shows potent antileukemia activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3775.

Abstract 3934: KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI)

Abstract Background: The murine double minute 2 (MDM2) oncoprotein is a key E3 ubiquitin ligase that degrades the tumor-suppressor p53. Reversible SMIs of the MDM2/p53 interaction have been developed to stabilize p53 and to induce apoptosis in wildtype p53 tumors. However, MDM2 SMIs induce a p53/MDM2 feedback loop, resulting in upregulation of MDM2 protein levels and p53 pathway inhibition, thus drastically limiting their biological activity and clinical application. MDM2 targeted protein degradation suppresses p53-dependent MDM2 protein feedback upregulation and is therefore expected to lead to a superior response compared to SMIs. Objectives: We discovered KT-253, a novel, highly potent heterobifunctional MDM2 degrader. We characterized the activity and molecular mechanism of KT-253 in wildtype p53 cell lines and xenograft models and demonstrated that KT-253 has superior activity compared to SMIs in these models. Methods: We characterized KT-253 in MDM2-dependent cell lines in vitro, as well as in in vivo mouse AML and ALL xenograft models. Methods included in vitro cell proliferation and apoptosis assays, gene expression profiling, and in vivo pharmacological studies. Results: KT-253 selectively depletes MDM2 protein levels with sub-nanomolar cellular potency. We demonstrated in the Acute Lymphoblastic Leukemia (ALL) cell line RS4;11 that KT-253 can overcome the p53-dependent upregulation of MDM2 protein levels as seen for reversible SMIs. In addition, even brief exposures to KT-253 in these cells could more potently stabilize p53 compared to SMIs, leading to apoptosis mediated through p53 target genes. The superior MDM2/p53 pathway inhibition and induction of apoptosis by KT-253 translates into a &amp;gt;200-fold stronger cell growth inhibition, compared to SMIs, across a panel of solid and hematological tumor cell lines. Using mouse xenograft models, we established an intermittent dosing schedule that drives anti-tumor efficacy. Importantly, a single dose of KT-253 at 1 mg/kg resulted in sustained tumor regression in the RS4;11 mouse xenograft model. In this model, KT-253 exposures correlated with induction of apoptotic p53 target genes and tumor growth inhibition. In addition, weekly administration of KT-253 significantly prolonged the survival in a disseminated Acute Myeloid Leukemia (AML) model when compared to vehicle treated animals. Conclusions: In summary, KT-253, our development candidate, has significantly improved potency relative to reversible SMIs leading to potent in vitro and in vivo efficacy that is superior to all clinically active agents. In addition, an intermittent dosing schedule of the highly potent KT-253 degrader can induce rapid apoptosis in MDM2-dependent cancer cells potentially leading to improved efficacy and safety profile. Citation Format: Yogesh Chutake, Michele Mayo, Dapeng Chen, Bradley Enerson, Patricia Cho, Jessica Filiatrault, Crystal Brown, Michael Placke, Madison Adams, Rahul Karnik, James Shaw, Yatao Shi, Dirk Walther, Alice McDonald, Frank Qi, Phillip Liu, Joseph D. Growney, Kirti Sharma, Duncan Walker, Stefanie Schalm, Juliet Williams, Matthew Weiss. KT-253, a highly potent and selective heterobifunctional MDM2 degrader for the treatment of wildtype p53 tumors with superior potency and differentiated biological activity compared to small molecule inhibitors (SMI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3934.

In Silico Analysis of L-Asparaginase Exposure on Acute Lymphoblastic Leukemia Cell Line RS 4: 11

Background The antineoplastic action of Asparaginase (ASNase) was connected to the inability of certain cancer cells to synthesise appropriate level of asparagine (asn). Due to the high affinity of clinically applicable ASNase for asn, asn is rapidly decreased in serum and extracellular fluids following intravenous or intramuscular injection. Tumor cells dependent on external Asn supply and unable of mitigating asn deficiency on their own may starve to death if exogenous asn depletion persists. Methods The current study focused on identifying Differentially Expressed Genes (DEGs) in Acute Lymphoblastic Leukemia (ALL) cell line RS 4;11. A complex biomolecular network for the DEGs was built and various tools were used to compute network parameters and identify drug targets. Results The biomolecular network comprised 109 DEGs. NOTCH1 and PIK3CA may be hub genes and require further research. Conclusion DEGs found could be employed as therapeutic targets for ALL tests or as leads for novel ALL treatments. The current study clarifies the L-ASNase’s exposure on sensitive ALL cell line; resistant cell lines may be investigated further in the future to obtain a greater understanding of ALL.

Characterization of the Impact of the MYBBP1A Gene and rs3809849 on Asparaginase Sensitivity and Cellular Functions

Aims: To investigate the role of MYBBP1A gene and rs3809849 in pancreatic cancer (PANC1) and lymphoblastic leukemia (NALM6) cell lines and their response to asparaginase treatment. Materials & methods: The authors applied CRISPR-Cas9 to produce MYBBP1A knock-out (KO) and rs3809849 knock-in (KI) cell lines. The authors also interrogated rs3809849's impact on PANC1 cells through allele-specific overexpression. Results: PANC1 MYBBP1A KO cells exhibited lower proliferation capacity (p≤0.05), higher asparaginase sensitivity (p=0.01), reduced colony-forming potential (p=0.001), cell cycle blockage in S phase, induction of apoptosis and remarkable morphology changes suggestive of an epithelial-mesenchymal transition. Overexpression of the wild-type(but not the mutant)allele ofMYBBP1A-rs3809849 in PANC1 cells increased asparaginase sensitivity. NALM6 MYBBP1A KO displayed resistance to asparaginase (p<0.0001), whereas no effect for rs3809849 KI was noted. Conclusions: MYBBP1A is important for regulating various cellular functions, and it plays, along with its rs3809849 polymorphism, a tissue-specific role in asparaginase treatment response.

The Cooperative Anti-Neoplastic Activity of Polyphenolic Phytochemicals on Human T-Cell Acute Lymphoblastic Leukemia Cell Line MOLT-4 In Vitro.

Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting pediatric patients. ALL treatment regimens with cytostatics manifest substantial toxicity and have reached the maximum of well-tolerated doses. One potential approach for improving treatment efficiency could be supplementation of the current regimen with naturally occurring phytochemicals with anti-cancer properties. Nutraceuticals such as quercetin, curcumin, resveratrol, and genistein have been studied in anti-cancer therapy, but their application is limited by their low bioavailability. However, their cooperative activity could potentially increase their efficiency at low, bioavailable doses. We studied their cooperative effect on the viability of a human ALL MOLT-4 cell line in vitro at the concentration considered to be in the bioavailable range in vivo. To analyze their potential side effect on the viability of non-tumor cells, we evaluated their toxicity on a normal human foreskin fibroblast cell line (BJ). In both cell lines, we also measured specific indicators of cell death, changes in cell membrane permeability (CMP), and mitochondrial membrane potential (MMP). Even at a low bioavailable concentration, genistein and curcumin decreased MOLT-4 viability, and their combination had a significant interactive effect. While resveratrol and quercetin did not affect MOLT-4 viability, together they enhanced the effect of the genistein/curcumin mix, significantly inhibiting MOLT-4 population growth in vitro. Moreover, the analyzed phytochemicals and their combinations did not affect the BJ cell line. In both cell lines, they induced a decrease in MMP and correlating CMP changes, but in non-tumor cells, both metabolic activity and cell membrane continuity were restored in time. (4) Conclusions: The results indicate that the interactive activity of analyzed phytochemicals can induce an anti-cancer effect on ALL cells without a significant effect on non-tumor cells. It implies that the application of the combinations of phytochemicals an anti-cancer treatment supplement could be worth further investigation regardless of their low bioavailability.