Leukemia Cell Line Research Articles

Targeted degradation of oncogenic BCR-ABL by silencing the gene of NEDD8 E3 ligase RAPSYN

Tyrosine kinase inhibitors have been the standard treatment for patients with Philadelphia chromosome-positive (Ph+) leukemia. However, a series of issues, including drug resistance, relapse and intolerance, are still an unmet medical need. Here, we report the targeted siRNA-based lipid nanoparticles in Ph+ leukemic cell lines for gene therapy of Ph+ leukemia, which specifically targets a recently identified NEDD8 E3 ligase RAPSYN in Ph+ leukemic cells to disrupt the neddylation of oncogenic BCR-ABL. To achieve the specificity for Ph+ leukemia therapy, a single-chain fragment variable region (scFv) of anti-CD79B monoclonal antibody was covalently conjugated on the surface of OA2-siRAPSYN lipid nanoparticles to generate the targeted lipid nanoparticles (scFv-OA2-siRAPSYN). Through effectively silencing RAPSYN gene in leukemic cell lines by the nanoparticles, BCR-ABL was remarkably degraded accompanied by the inhibition of proliferation and the promotion of apoptosis. The specific targeting, therapeutic effects and systemic safety were further evaluated and demonstrated in cell line-derived mouse models. The present study has not only addressed the clinical need of Ph+ leukemia, but also enabled gene therapy against a less druggable target.Graphical

Deep PIM kinase substrate profiling reveals new rational co-therapeutic strategies for acute myeloid leukemia.

Provirus integration site for Moloney murine leukemia virus (PIM) family serine/threonine kinases perform pro-tumorigenic functions in hematologic malignancies and solid tumors by phosphorylating substrates involved in tumor metabolism, cell survival, metastasis, inflammation, and immune cell invasion. However, a comprehensive understanding of PIM kinase functions is currently lacking. Multiple small molecule PIM kinase inhibitors are currently being evaluated as co-therapeutics in cancer patients. To further illuminate PIM kinase functions in cancer, we deeply profiled PIM1 substrates using the reverse in-gel kinase assay to identify downstream cellular processes targetable with small molecules. Pathway analyses of putative PIM substrates nominated RNA splicing and rRNA processing as PIM-regulated cellular processes. PIM inhibition elicited reproducible splicing changes in PIM-inhibitor-responsive acute myeloid leukemia (AML) cell lines. PIM inhibitors synergized with splicing modulators targeting splicing factor 3b subunit 1 (SF3B1) and serine-arginine protein kinase 1 (SRPK1) to kill AML cells. PIM inhibition also altered rRNA processing, and PIM inhibitors synergized with an RNA polymerase I inhibitor to kill AML cells and block AML tumor growth. These data demonstrate that deep kinase substrate knowledge can illuminate unappreciated kinase functions, nominating synergistic co-therapeutic strategies. This approach may expand the co-therapeutic armamentarium to overcome kinase-inhibitor resistant disease that limits durable responses in malignant disease.

Effective delivery of anti-PD-L1 siRNA with human heavy chain ferritin (HFn) in acute myeloid leukemia cell lines.

Because of the high biocompatibility, self-assembly capability, and CD71-mediated endocytosis, using human heavy chain ferritin (HFn) as a nanocarrier would greatly increase therapeutic effectiveness and reduce possible adverse events. Anti-PD-L1 siRNA can downregulate the level of PD-L1 on tumor cells, resulting in the activation of effector T cells against leukemia. Therefore, this study aimed to produce the tumor-targeting siPD-L1/HFn nanocarrier. Briefly, the HFn coding sequence was cloned into a pET-28a, and the constructed expression plasmid was subsequently transformed into E. coli BL21. After induction of Isopropyl β-D-1-thiogalactopyranoside (IPTG), HFn was purified with Ni-affinity chromatography and dialyzed against PBS. The protein characteristics were analyzed using SDS-PAGE, Western Blot, and Dynamic light scattering (DLS). The final concentration was assessed using the Bicinchoninic acid (BCA) assay. The encapsulation was performed using the standard pH system. The treatment effects of siPD-L1/HFn were carried out on HL-60 and K-562 cancer cell lines. The RT-PCR was used to determine the mRNA expression of PD-L1. The biocompatibility and excretion of siPD-L1/HFn have also been evaluated. The expression and purity of HFn were well verified through SDS-PAGE, WB, and DLS. RT-PCR analyses also showed significant siRNA-mediated PD-L1 silencing in both HL-60 and K-562 cells. Our study suggested a promising approach for siRNA delivery. This efficient delivery system can pave the way for the co-delivery of siRNAs and multiple chemotherapies to address the emerging needs of cancer combination therapy.

A new therapeutic perspective: Erastin inhibits tumor progression by driving ferroptosis in myelodysplastic syndromes.

Ferroptosis is a recently identified and evolutionarily conserved form of programmed cell death. This process is initiated by an imbalance in iron metabolism, leading to an overload of ferrous ions. These ions promote lipid peroxidation in the cell membrane through the Fenton reaction. As the cell's antioxidant defenses become overwhelmed, a fatal buildup of reactive oxygen species (ROS) occurs, resulting in the rupture of the plasma membrane. Ferroptosis is implicated in conditions such as ischemia-reperfusion injuries and a range of cancers. In our research, we explored ferroptosis in myelodysplastic syndromes (MDS) by measuring iron levels, transferrin receptor expression, and glutathione peroxidase 4 (GPX4) mRNA. Our findings revealed that MDS patients had significantly higher Fe2+ levels in CD33+ cells and increased transferrin receptor mRNA compared to healthy individuals. GPX4 expression was also higher in MDS but not statistically significant. To investigate potential treatments for myeloid hematological diseases through ferroptosis induction, we treated the myelodysplastic syndrome cell line (SKM-1) and two myeloid leukemia cell lines (KG-1 and K562) with erastin, an iron transfer inducer. We observed that erastin treatment led to glutathione depletion, reduced GPX4 activity, and increased ROS, culminating in cell death by ferroptosis. Furthermore, combining erastin with azacitidine demonstrated a synergistic effect on MDS and leukemia cell lines, suggesting a promising approach for treating these hematological conditions with this drug combination. Our experiments confirm erastin's ability to induce ferroptosis in MDS and highlight its potential synergistic use with azacitidine for treatment.

Harnessing molecular hybridization approach to discover novel quinoline EGFR-TK inhibitors for cancer treatment.

Aim: Using molecular hybridization approach, novel 18 quinoline derivatives (6a-11) were designed and synthesized as EGFR-TK inhibitors. Materials & methods: The antiproliferative activity was assessed against breast (MCF-7), leukemia (HL-60) and lung (A549) cancer cell lines. Moreover, the most active quinoline derivatives (6d and 8b) were further investigated for their potential as EGFR-TK inhibitors. In addition, cell cycle analysis and apoptosis induction activity were conducted. Results: A considerable cytotoxic activity was attained with IC50 values spanning from 0.06 to 1.12μM. Besides, the quinoline derivatives 6d and 8b displayed potent inhibitory activity against EFGR with IC50 values of 0.18 and 0.08μM, respectively. Conclusion: Accordingly, the afforded quinoline derivatives can be used as promising lead anticancer candidates for future optimization.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2-positive lymphoid malignancies irrespective of BIM status

Venetoclax, a BCL2 inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large B-cell lymphomas (LBCL), but remissions were generally short, which calls for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples we demonstrated strong synergy between venetoclax and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments, and studies on clones with knockout of expression, or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired venetoclax resistance. Of note, the venetoclax and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the pro-apoptotic BCL2L11/BIM, and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in venetoclax resistance. Immunoprecipitation experiments further suggested that the pro-apoptotic effector BAX belongs to principal mediators of the venetoclax and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new pro-apoptotic combination was confirmed in vivo on a panel of 9 PDX models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and DLBCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days ON / 3 days OFF treatment regimen, which retained the desired anti-tumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2-positive hematologic malignancies irrespective of the BCL1L11/BIM status.

[Study on the inhibitory and pro-apoptotic effects of different concentrations of total tanshinone alone and in combination with tyrosine kinase inhibitors on human myeloid leukemia cell lines].

Objective: To explore the effect and investigate the molecular mechanism of different concentrations of total tanshinones alone and in combination with tyrosine kinase inhibitors (TKIs) on the proliferation inhibition and apoptosis of human myeloid leukemia cell lines. Methods: K562 and Kasumi-1 cell lines were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the TKIs-resistant strain K562/T315I cell line was constructed in Molecular Medicine Research Center, Beijing Lu Daopei Institute of Hematology. Logarithmic growth phase cells were taken and divided into intervention groups with total tanshinone of 0, 2.19, 4.38, 8.75, 17.50 and 35.00 μg/ml intervention groups, which were inoculated in 96-well plates at a density of 1×104 cells/well and exposed to the drug for 24 h, and a control group treated with dimethyl sulfoxide was also set up simultaneously. All experiments were repeated independently 3-5 times. The proliferative activity of the cells was assessed using the CCK-8 assay, the apoptotic rates were measured by flow cytometry, and the expression levels of apoptosis-regulating proteins Bcl-2 and Bax were analyzed by Western blotting. The cell lines treated and untreated with total tanshinone were subjected to transcriptome sequencing and gene set enrichment analysis to identify differentially expressed genes. Results: The half-inhibitory concentration (IC50) values of 8.75 μg/ml total tanshinone at 24 h for K562, K562/T315I and Kasumi-1 cells were (4.11±0.02), (4.95±0.04) and (3.98±0.01) μg/ml, respectively. When combined with 0.25 μmol/L imatinib, 8.75 μg/ml total tanshinone could enhance the induction of apoptosis effects on K562 and K562/T315I cell lines. After being treated with 4.38, 8.75, and 17.50 μg/ml of total tanshinone for 24 h, compared with the control group, total tanshinone upregulated the expression level of Bax protein, downregulated the expression level of Bcl-2 protein, and decreased the Bcl-2/Bax ratio (all P<0.05). Total tanshinone inhibited the proliferation-related signaling pathway and DNA damage repair pathway of myeloid leukemia cell lines, and activated the signaling pathway that induces apoptosis in leukemia cells. Conclusion: Different concentrations of total tanshinoneinhibites proliferation and promote apoptosis in K562, Kasumi-1 and TKIs-resistant K562/T315I cell lines, and further enhance the anti-leukemic effect when combined with TKIs.

Synthesis, design, and antiproliferative evaluation of 6-(N-Substituted-methyl)pyrazolo[3,4-d]pyrimidines as the potent anti-leukemia agents

Pyrazolopyrimidine derivatives, including pyrazolopyrimidines, 6-aminopyrazolopyrimidines, 6-[(formyloxy)methyl]pyrazolopyrimidines, 6-(hydroxymethyl)pyrazolopyrimidine, and 6-(aminomethyl)pyrazolopyrimidines have been successfully prepared and tested against NCI-H226, NPC-TW01, and Jurkat cancer cell lines. Among the tested pyrazolopyrimidine compounds, we found 6-aminopyrazolopyrimidines and 6-(aminomethyl)pyrazolopyrimidines with essential o-ClPh or p-ClPh substituted moieties on N-1 pyrazole ring exhibited the best IC50 inhibition activity for Jurkat cells. Furthermore, optimization of the SAR study on the C-6 position of pyrazolopyrimidine ring demonstrated that 6-(N-substituted-methyl)pyrazolopyrimidines 17b, 17d, and 19d possessed the significant IC50 inhibitory activity for the different leukemia cell lines, especially for Jurkat, K-562, and HL-60. On the other hand, further SAR inhibition and docking model studies revealed that compound 19d, which has a 3-(1H-imidazol-1-yl)propan-1-amino side-chain on the C-6 position, was able to form four hydrogen bonds with residues Ala226, Leu152, and Glu194 and specifically extended into the P1 pocket subsite with Aurora A, resulting in improved inhibitory activity almost similar to SNS-314. To explore the anti-cancer mechanism, compound 19d was measured by Western blot analysis in Jurkat T-cells, however, it showed non-responsibility to Aurora B. For the further structural modifications on the lateral chain of compound 19d, compounds 24 with longer lateral chain were designed and synthesized for testing leukemia cell lines. However, compounds 24 was significantly decrease inhibition potency against leukemia cell lines. Based on the in-vitro results, compounds 17b and 19d could be considered to be the best potential lead drug in our study for the development of new and effective therapies for leukemia treatment. On the other hand, the DHFR inhibition results indicated compound 19d possessed good inhibitory activity and better than the reported naphthalene derivative. Through further comparisons of the model superposition of three-dimensional (3D) conformations in DHFR, compound 19d presented a similar structural alignment to Methotrexate and the reported naphthalene derivative and led to similar drug-like functional relationships. As a results, compound 19d would be a potential DHFR inhibitor for anti-leukemia drug candidate.

Thiazole Derivatives as RORγt Inhibitors: Synthesis, Biological Evaluation, and Docking Analysis

Background: Cancer is a serious concern in developing countries. Due to various worldwide impacts, it has surpassed coronary disorders becoming the leading cause of mortality. In cancer progression, the involvement of RORs (retinoic acid receptor-related orphan receptors) has gained attention in the search for versatile agents. Thiazole has the potential to be used as a scaffold in the development of anticancer drugs. Objective: This work’s major goal was to design and synthesize promising anticancer candidates. Methods: Docking investigations concerning the RORγt receptor were utilized in the design of the compounds. A series of novel thiazole derivatives was synthesized employing Hantsch synthesis. Melting points, TLC development, and spectral analyses were considered for the synthesized compounds' characterization. Corresponding spectra were analyzed and determined to be consistent with the assigned structure. On 60 human cancer cell lines, selected compounds were investigated in vitro by NCI (National Cancer Institute), USA, for anticancer action. Results: Varying ratios of anticancer potential were presented by the compounds in in vitro studies. Compound 3c was determined to be the most active compound in the series, with a -15.03 mean growth percent. In silico study revealed that the synthesized compounds could have anticancer possibilities. In the targeted pdb id (6q7a) binding site, compound 3c demonstrated excellent docking scores in molecular docking analyses, supporting its ability to be utilized as a lead compound in rational drug design. Conclusion: Compounds with satisfactory docking scores within the binding pocket of chosen pdb id (6q7a) validated their suitability for usage as lead compounds in rational drug design. Outcomes of in silico investigations were in agreement with the findings of the cytotoxicity experiments. Leukemia, renal, breast, and lung cancer cell lines were most sensitive to the substance-treated tumor cells. Electronwithdrawing groups were essential for antiproliferative action, according to the findings.

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

Expression of transcriptional repressors of ZBTB genes in acute lymphoblastic leukemia cell lines treated with prednisolone

In vitro anti-leukemic effect of Wharton's jelly derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have the ability to self-renew and are multi-potent. They are a primary candidate for cell-based therapy due to their potential anti-cancer effects. The aim of this study was to evaluate the in vitro anti-leukemic effect of Wharton's Jelly-derived MSC (WJ-MSC) on the leukemic cell lines K562 and HL-60. In this present study, WJ-MSCs were isolated from human umbilical cord. The cells were incubated according to the standard culture conditions and characterized by flow cytometry. For experiments, WJ-MSC and leukemic cells were incubated in the direct co-culture at a ratio of 1:5 (leukemia cells: WJ-MSC). HUVEC cells were used as a non-cancerous cell line model. The apoptotic effect of WJ-MSCs on the cell lines was analyzed using Annexin V/PI apoptosis assay. After the direct co-culture of WJ-MSCs on leukemic cell lines, we observed anti-leukemic effects by inducing apoptosis. We had two groups of determination apoptosis with and without WJ-MSCs for all cell lines. Increased apoptosis rates were observed in K562 and HL-60 cell lines, whereas the apoptosis rates in HUVEC cells were low. MSCs are known to inhibit the growth of tumors of both hematopoietic and non-hematopoietic origin in vitro. In our study, WJ-MSC treatment strongly inhibited the viability of HL-60 and K562 and induced apoptosis. Our results also provided new insights into the inhibition of tumor growth by WJ-MSCs in vitro. In the future, WJ-MSCs could be used to inhibit cancer cells in clinical applications.

Herpesvirus Entry Mediator as an Immune Checkpoint Target and a Potential Prognostic Biomarker in Myeloid and Lymphoid Leukemia

Herpesvirus entry mediator (HVEM) is a molecular switch that can modulate immune responses against cancer. The significance of HVEM as an immune checkpoint target and a potential prognostic biomarker in malignancies is still controversial. This study aims to determine whether HVEM is an immune checkpoint target with inhibitory effects on anti-tumor CD4+ T cell responses in vitro and whether HVEM gene expression is dysregulated in patients with acute lymphocytic leukemia (ALL). HVEM gene expression in tumor cell lines and peripheral blood mononuclear cells (PBMCs) from ALL patients and healthy controls was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Tumor cells were left untreated (control) or were treated with an HVEM blocker before co-culturing with CD4+ T cells in vitro in a carboxyfluorescein succinimidyl ester (CFSE)-dependent proliferation assay. HVEM expression was upregulated in the chronic myelogenous leukemia cell line (K562) (FC = 376.3, p = 0.086) compared with normal embryonic kidney cells (Hek293). CD4+ T cell proliferation was significantly increased in the HVEM blocker-treated K562 cells (p = 0.0033). Significant HVEM differences were detected in ALL PBMCs compared with the controls, and these were associated with newly diagnosed ALL (p = 0.0011) and relapsed/refractory (p = 0.0051) B cell ALL (p = 0.0039) patients. A significant differentiation between malignant ALL and the controls was observed in a receiver operating characteristic (ROC) curve analysis with AUC = 0.78 ± 0.092 (p = 0.014). These results indicate that HVEM is an inhibitory molecule that may serve as a target for immunotherapy and a potential ALL biomarker.

Novel guanidine derivatives targeting leukemia as selective Src/Abl dual inhibitors: Design, synthesis and anti-proliferative activity

A new series of benzene-sulfonamide derivatives 3a-i was designed and synthesized via the reaction of N-(pyrimidin-2-yl)cyanamides 1a-i with sulfamethazine sodium salt 2 as dual Src/Abl inhibitors. Spectral data IR, 1H-, 13C- NMR and elemental analyses were used to confirm the structures of all the newly synthesized compounds 3a-i and 4a-i. Crucially, we screened all the synthesized compounds 3a-i against NCI 60 cancer cell lines. Among all, compound 3b was the most potent, with IC50 of 0.018 μM for normoxia, and 0.001 μM for hypoxia, compared to staurosporine against HL-60 leukemia cell line. To verify the selectivity of this derivative, it was assessed against a panel of tyrosine kinase EGFR, VEGFR-2, B-raf, ERK, CK1, p38-MAPK, Src and Abl enzymes. Results revealed that compound 3b can effectively and selectively inhibit Src/Abl with IC500.25 μM and Abl inhibitory activity with IC500.08 μM, respectively, and was found to be more potent on these enzymes than other kinases that showed the following results: EGFR IC500.31 μM, VEGFR-2 IC500.68 μM, B-raf IC500.33 μM, ERK IC501.41 μM, CK1 IC500.29 μM and p38-MAPK IC500.38 μM. Moreover, cell cycle analysis and apoptosis performed to compound 3b against HL-60 suggesting its antiproliferative activity through Src/Abl inhibition. Finally, molecular docking studies and physicochemical properties prediction for compounds 3b, 3c, and 3 h were carried out to investigate their biological activities and clarify their bioavailability.

Variable characteristics overlooked in human K-562 leukemia cell lines with a common signature

K-562 is a well-known in vitro cellular model that represents human leukemia cell lines. Although the K-562 cells have been extensively characterized, there are inconsistencies in the data across publications, showing the presence of multiple K-562 cell lines. This suggests that analyzing a single K-562 cell line is insufficient to provide reliable reference data. In this study, we compared three K-562 cell lines with different IDs (RCB0027, RCB1635, and RCB1897) to investigate the fundamental characteristics of K-562 cells. Amplifications of the BCR-ABL1 fusion gene and at 13q31 were detected in all three cell lines, whereas each genome exhibited distinctive features of sequence variants and loss of heterozygosity. This implies that each K-562 cell line can be characterized by common and unique features through a comparison of multiple K-562 cell lines. Variations in transcriptome profiles and hemoglobin synthesis were also observed among the three cell lines, indicating that they should be considered sublines that have diverged from the common ancestral K-562 despite no changes from the original cell name. This leads to unintentional differences in genotypes and/or phenotypes among cell lines that share the same name. These data show that characterizing a single K-562 cell line does not necessarily provide data that are applicable to other K-562 cells. In this context, it is essential to modify cell names in accordance with changes in characteristics during cell culture. Furthermore, our data could serve as a reference for evaluating other K-562 sublines, facilitating the discovery of new K-562 sublines with distinct characteristics. This approach results in the accumulation of K-562 sublines with diverged characteristics and expands the options available, which may help in selecting the most suitable K-562 subline for each experiment.

(2,6-Dimethylphenyl)arsonic Acid Induces Apoptosis through the Mitochondrial Pathway, Downregulates XIAP, and Overcomes Multidrug Resistance to Cytostatic Drugs in Leukemia and Lymphoma Cells In Vitro.

Cancer treatment is greatly challenged by drug resistance, highlighting the need for novel drug discoveries. Here, we investigated novel organoarsenic compounds regarding their resistance-breaking and apoptosis-inducing properties in leukemia and lymphoma. Notably, the compound (2,6-dimethylphenyl)arsonic acid (As2) demonstrated significant inhibition of cell proliferation and induction of apoptosis in leukemia and lymphoma cells while sparing healthy leukocytes. As2 reached half of its maximum activity (AC50) against leukemia cells at around 6.3 µM. Further experiments showed that As2 overcomes multidrug resistance and sensitizes drug-resistant leukemia and lymphoma cell lines to treatments with the common cytostatic drugs vincristine, daunorubicin, and cytarabine at low micromolar concentrations. Mechanistic investigations of As2-mediated apoptosis involving FADD (FAS-associated death domain)-deficient or Smac (second mitochondria-derived activator of caspases)/DIABLO (direct IAP binding protein with low pI)-overexpressing cell lines, western blot analysis of caspase-9 cleavage, and measurements of mitochondrial membrane integrity identified the mitochondrial apoptosis pathway as the main mode of action. Downregulation of XIAP (x-linked inhibitor of apoptosis protein) and apoptosis induction independent of Bcl-2 (B-cell lymphoma 2) and caspase-3 expression levels suggest the activation of additional apoptosis-promoting mechanisms. Due to the selective apoptosis induction, the synergistic effects with common anti-cancer drugs, and the ability to overcome multidrug resistance in vitro, As2 represents a promising candidate for further preclinical investigations with respect to refractory malignancies.

TurboID-Based IRE1 Interactome Reveals Participants of the Endoplasmic Reticulum-Associated Protein Degradation Machinery in the Human Mast Cell Leukemia Cell Line HMC-1.2.

The unfolded protein response is an intricate system of sensor proteins in the endoplasmic reticulum (ER) that recognizes misfolded proteins and transmits information via transcription factors to either regain proteostasis or, depending on the severity, to induce apoptosis. The main transmembrane sensor is IRE1α, which contains cytoplasmic kinase and RNase domains relevant for its activation and the mRNA splicing of the transcription factor XBP1. Mast cell leukemia (MCL) is a severe form of systemic mastocytosis. The inhibition of IRE1α in the MCL cell line HMC-1.2 has anti-proliferative and pro-apoptotic effects, motivating us to elucidate the IRE1α interactors/regulators in HMC-1.2 cells. Therefore, the TurboID proximity labeling technique combined with MS analysis was applied. Gene Ontology and pathway enrichment analyses revealed that the majority of the enriched proteins are involved in vesicle-mediated transport, protein stabilization, and ubiquitin-dependent ER-associated protein degradation pathways. In particular, the AAA ATPase VCP and the oncoprotein MTDH as IRE1α-interacting proteins caught our interest for further analyses. The pharmacological inhibition of VCP activity resulted in the increased stability of IRE1α and MTDH as well as the activation of IRE1α. The interaction of VCP with both IRE1α and MTDH was dependent on ubiquitination. Moreover, MTDH stability was reduced in IRE1α-knockout cells. Hence, pharmacological manipulation of IRE1α-MTDH-VCP complex(es) might enable the treatment of MCL.

Porphyrin overdrive rewires cancer cell metabolism.

All cancer cells reprogram metabolism to support aberrant growth. Here, we report that cancer cells employ and depend on imbalanced and dynamic heme metabolic pathways, to accumulate heme intermediates, that is, porphyrins. We coined this essential metabolic rewiring "porphyrin overdrive" and determined that it is cancer-essential and cancer-specific. Among the major drivers are genes encoding mid-step enzymes governing the production of heme intermediates. CRISPR/Cas9 editing to engineer leukemia cell lines with impaired heme biosynthetic steps confirmed our whole-genome data analyses that porphyrin overdrive is linked to oncogenic states and cellular differentiation. Although porphyrin overdrive is absent in differentiated cells or somatic stem cells, it is present in patient-derived tumor progenitor cells, demonstrated by single-cell RNAseq, and in early embryogenesis. In conclusion, we identified a dependence of cancer cells on non-homeostatic heme metabolism, and we targeted this cancer metabolic vulnerability with a novel "bait-and-kill" strategy to eradicate malignant cells.

INA03, a potent transferrin-competitive antibody-drug conjugate against CD71, for a safer acute leukemia treatment.

Innovative strategies to enhance efficacy and overcome drug resistance in hematologic cancers such as antibody-drug conjugates (ADCs) have shifted the paradigm of conventional care by delivering promising outcomes in cancer therapies with a significant reduction in the risk of relapse. The transferrin receptor 1, CD71, known to be overexpressed in malignant cells, is considered a potent anti-tumoral target. Therefore, we have developed an anti-CD71 ADC, INA03, a humanized antibody conjugated to the monomethyl auristatin E (MMAE) through a 3-arylpropiolonitrile-valine-citruline linker. In this study, we investigated both potency and safety of INA03, in competition with transferrin (Tf), the CD71's natural ligand, as a novel strategy to specifically target highly proliferative cells. The high expression of CD71 was confirmed on different leukemic cell lines, allowing INA03 to bind efficiently. Subsequently, INA03 rapidly internalizes into lysosomal compartments, where its cytotoxic drug is released following cathepsin-B cleavage. Downregulating CD71 expression using shRNA highlighted that INA03-induced cell death was dependent on CD71 density at the cell surface. INA03 intravenous treatment in acute leukemia mouse models significantly reduced tumor burden, increased mice survival and showed no residual disease compared to conventional chemotherapies. Since INA03 competes with the human Tf, a double knock-in (hCD71/hTf) competent mouse model was generated to mimic human pharmacokinetics and pharmacodynamics. INA03 administration in hCD71/hTf mice did not reveal, even at high doses, any improper toxicities. Hence, these data demonstrate promising pre-clinical efficacy and safety of INA03 and support its development as a novel acute leukemia treatment.

UBE2C promotes the proliferation of acute myeloid leukemia cells through PI3K/AKT activation

This study aims to investigate the role and mechanism of tubiquitin-conjugating enzyme E2 C (UBE2C) in acute myeloid leukemia (AML). Initially, UBE2C expression in leukemia was analyzed using the Cancer Genome Atlas database. Further, we silenced UBE2C expression using small-hairpin RNA (sh-RNA). UBE2C expression was detected via the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis. Apoptotic events and reactive oxygen species (ROS) levels were detected by flow cytometry. A xenograft model of leukemia cells were established, and the protein levels of UBE2C, KI-67, and cleaved-caspase 3 were detected by immunohistochemistry. We reported an overexpression of UBE2C in leukemia patients and cell lines (HL60, THP-1, U937, and KG-1 cells). Moreover, a high expression level of UBE2C was correlated with a dismal prognosis in AML patients. UBE2C knockdown inhibited the viability and promoted apoptosis in AML cells by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Furthermore, UBE2C knockdown increased cellular Fe2+ and ROS levels, and enhanced erastin-induced ferroptosis in a proteasome-dependent manner. UBE2C knockdown also suppressed the tumor formation of AML cells in the mouse model. In summary, our findings suggest that UBE2C overexpression promotes the proliferation and inhibits ferroptosis in AML cells by activating the PI3K/AKT pathway.

Synthesis, Biological Evaluation, Molecular Docking and ADME Profiling of Methylpiperidin‐4‐ylphenyl‐nicotinamide Derivatives

AbstractIn this paper, we designed, synthesized, and biologically screened a series of methyl‐piperidine‐phenyl‐nicotinamide derivatives with the aim of novel derivatives as effective anticancer agents. Compounds (9 a), (9 e) and (9 k) exhibited potent anticancer activity with an IC50 value of 5.9 μM, 5.9 μM and 5.0 μM, respectively using MV411 (Acute Myeloid Leukemia) and K562 (Chronic Myeloid Leukemia) cell line. Our docking studies show good binding affinity of these compounds for ABL1 kinase. Furthermore, these compounds exhibited a favourable in silico ADME profile with good solubility and low clearance in Human Liver Microsomes (HLM). These derivatives represent a promising approach to initiate the development of new anticancer drugs in oncology programs.