Proliferation Of Leukemia Cell Lines Research Articles

Preclinical Activity of Novel TGF Beta Receptor I Kinase Inhibitors IOA-359 and IOA-360 for Treatment of Anemia in MDS/AML

Background: Overactivation of the Transforming growth factor beta (TGF-β) superfamily has been associated with bone marrow failure in MDS. TGF-β binds to set of receptors that include the TGF-β receptor I kinase (also known as ALK5), that in turn phosphorylates and activates the downstream SMAD2/3 proteins. Activation of SMAD2/3 transcription factors has been shown to occur in MDS and is associated with anemia. Thus, we wanted to evaluate the preclinical efficacy of novel, clinic-ready, TGF-b receptor I kinase small molecule inhibitors IOA-359 and IOA-360 in MDS models. Results: Using the TREEspot™ SCAN Max Kinase Panel of 403 kinases, IOA-359 and IOA-360 were found to be highly selective for ALK5. Both, IOA-359 and IOA-360 have a blocking activity against ALK5 similar to clinical stage ALK5 inhibitors. To evaluate the efficacy of IOA-359 and IOA-360, we first used HEK 293- Blue-TGF-β cells that stably overexpresses a SMAD inducible secreted embryonic alkaline phosphatase (SEAP) reporter. Both IOA-359 and IOA-360 were able to significantly inhibit TGF-β driven reporter at nanomolar doses ( Fig1A). Next, we showed that both ALK5 inhibitors were able to block TGF-β-mediated SMAD2/3 downstream phosphorylation/activation in a dose dependent manner in MDS and leukemic cell lines. Functionally, the ALK5 inhibitors did not lead to any increase in viability or proliferation in leukemic cell lines. We next tested the efficacy of the novel ALK5 inhibitors in primary patient samples from MDS and AML (N=8). Treatment with both ALK5 inhibitors led to increase in erythroid colony formation in majority of MDS samples in clonogenic assays. Colonies were picked up and examined for erythroid and myeloid differentiation by FACS analysis. We observed that ALK5 inhibition led to increase in Glycophorin A and CD71 expression suggesting enhanced erythroid differentiation ( Fig1B). Myeloid differentiation effects were modest in comparison. Furthermore, in selected samples, addition of Luspatercept led to greater maturation of erythrocytes. Treatment of healthy CD34 + hematopoietic stem and progenitors did not lead to significant changes in differentiation. Lastly, to evaluate downstream effects of ALK5 inhibition, we performed RNAseq analysis in MDS cells. Inhibition with ALK5 inhibitors led to numerous changes in genes affecting cellular differentiation, validating functional changes observed by us. Conclusion: Our current results support the preclinical in vitro efficacy of ALK5 inhibitors IOA-359 and IOA-360 alone and in combination with Luspartercept, highlighting their potential for further development and clinical testing in MDS/AML.

Impact of rhG-CSF on Clinical Efficacy and Immune Cell Subsets after Initial Induction Chemotherapy in AML.

The impact of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in acute myeloid leukemia (AML) is still controversial. The purpose of this study is to explore the impact of rhG-CSF administration on clinical efficacy and immune cell subsets after initial induction chemotherapy in AML. The clinical efficacy and immune cell subsets were compared in the newly diagnosed patients with AML according to whether rhG-CSF was used after initial induction chemotherapy. Next, rhG-CSF stimulation experi-ments on leukemia cell lines and primary leukemia blasts were performed in vitro. There was no statistical difference between control group and rhG-CSF therapy group in complete remission rate and relapse free survival. The duration of agranulocytosis was significantly shortened in rhG-CSF therapy group compared with control group. The percentage of circulating monocytic myeloid-derived suppressor cells (M-MDSCs) and regulatory T cells (Tregs) were significantly increased after the administration of rhG-CSF. Furthermore, it was found that rhG-CSF did not promote the proliferation of leukemia cell lines and primary leukemia blasts, but increased the proportion of M-MDSCs and Tregs in vitro. Administration of rhG-CSF after initial induction therapy of AML does not affect the clinical remission and relapse rate, but reduces the duration of agranulocytosis and increases the proportion of M-MDSCs and Tregs.

Phase 1 Results of the First-in-Class CXCR1/2 Inhibitor SX-682 in Patients with Hypomethylating Agent Failure Myelodysplastic Syndromes

Background: SX-682 is an oral dual allosteric inhibitor of the CXCR1 and CXCR2 (CXCR1/2) chemokine receptors, a signaling pathway involved in the recruitment of immunosuppressive MDSCs and autocrine growth of disease-initiating leukemic stem cells (LSCs) in AML and myelodysplastic syndromes (MDS). CXCR2 overexpression is predictive of transfusion dependence in MDS. CXCR2 inhibition reduces proliferation of leukemic cell lines and primary MDS/AML patient (pt) samples in vitro and impairs leukemic growth in an in vivo AML animal model. CXCR2 is also involved in normal neutrophil egress from the bone marrow and thus absolute neutrophil count (ANC) serves as a pharmacodynamic (PD) marker for CXCR1/2 inhibition by SX-682. In pts with normal bone marrow, 25-400 mg SX-682 twice-daily (BID) dose-dependently reduces ANC, with maximum ANC reduction (-66% of baseline) obtained at >150 mg BID, consistent with maximum CXCR1/2 inhibition above this dose. ANC rapidly (24 hrs) normalizes with dose-holding. This NHLBI funded trial (HL142389, NCT04245397) explored the safety and efficacy of SX-682 monotherapy in pts with hypomethylating agent (HMA) failure MDS. Methods: This Phase 1 dose-escalation with expansion study treated five cohorts of HMA failure MDS pts with escalating 25-400 mg BID doses of SX-682 in six continuous 28-day cycles with responding pts continuing treatment. Steady-state pharmacokinetics (PK) were obtained. Response assessment was at the end of cycles 1, 3 and 6 and every third cycle thereafter using IWG criteria (Cheson 2006). This interim analysis after dose-escalation to select the expansion dose was pre-specified. Results:17 MDS pts received SX-682 (median 1 prior therapies; range 1-4) with 6 pts in the 200 mg cohort, 3 pts in each of the 25, 50 and 100 cohorts and 2 pts in the 400 mg cohort (Table). Median age was 76, and pts were low (n=1), intermediate-1 (n=10), intermediate-2 (n=5) and high (n=1) risk by the International Prognostic Scoring System (IPSS). All pts were transfusion-dependent and all had failed prior HMA and 24% lenalidomide. Steady-state PK exhibits increasing drug plasma concentration with dose comparable to pts with solid tumors. The ANC PD marker exhibited a dose-dependent decrease from baseline (ΔANC) that plateaued in the 200 and 400 mg dose cohorts consistent with maximum receptor inhibition (Table). Marrow MDSCs and LSCs were reduced after initiation of SX-682. SX-682 was well-tolerated with no maximally tolerated dose and no pt discontinued treatment for adverse events. Treatment-emergent adverse events > grade 3 were most common in the 200 and 400 mg dose cohorts, and those related to neutrophils are consistent with being related to SX-682. Effects on neutrophils reversed on cessation of drug dosing. There was a dose-dependent increase in overall response rate (ORR) from 0% at 25 mg BID to 50% at 200 mg BID consistent with the observed clinical benefit being related to CXCR1/2 inhibition by SX-682 (Table). Four of the five responders had mutant genes (MGs) involving splicing factors (60% of responders; SF3B1, SRSF2) and/or epigenetic regulation (60% of responders; TET2, ASXL1, IDH2). Across all dose cohorts, 8 of 17 (47%) pts had a reduction in marrow blasts after initiating SX-682 (Figure). The 200 mg dose yielded the most rapid and deep reduction in blasts with 2 of 6 pts (33%) achieving a marrow complete remission (mCR) in keeping with this dose providing maximum receptor inhibition. There was hematologic improvement (HI) in 3 pts with one having a durable near-CR response (on SX-682 >500 days) consisting of trilineage HI (hemoglobin > 10 g/dL) and transfusion independence for over 4 months (pt required 16 transfusions in the 7 weeks before starting SX-682 therapy). Based on ANC PD, ORR and marrow blast responses, the 200 mg BID dose was selected for the expansion phase of the trial. Conclusions: SX-682 is a novel therapy in HMA failure MDS with dual activity against LSCs and MDSCs. SX-682 was well tolerated with encouraging efficacy with a biologically effective oral dose selected at 200 mg BID. An expansion cohort in both lower and higher risk MDS pts is ongoing and will additionally include HMA naïve patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Screening of proliferation related lncRNAs in leukemia cell lines by lentivirus shRNA library combined with second-generation sequencing

Long non-coding RNA (lncRNA) has become an important regulator of many cellular processes, including cell proliferation. Although studies have shown that a variety of lncRNAs play an important role in the occurrence and development of hematopoietic malignancies, a more comprehensive and unbiased method to study the function of lncRNAs in leukemia cell lines is lacking. Here, we used short hairpin RNA (shRNA) library combined with high-throughput sequencing to screen lncRNAs that may affect the proliferation of leukemia cell lines, and identified lncRNA C20orf204-203 among 74 candidate lncRNAs in this study. Further experiments showed that C20orf204-203 was localized in the cytoplasm in both K562 and THP-1 cell lines. C20orf204-203 knockdown decreased the proliferation of K562 and THP-1 cell lines accompanied with the increased proportion of early apoptotic cells. We observed the increased mRNA level of BAD gene while decreased protein level of TP53 and BCL2. The expression of Caspase 3 decreased and Caspase 3-cleaved protein increased in THP-1 cell line. However, their changes were inconsistent in the two cell lines. Our experimental results showed that knockdown of lncRNA C20orf204-203 in leukemia cell lines affected cell proliferation although the mechanism of action in different cell lines may differ. Importantly, our research demonstrated the feasibility of using shRNA library combined with high-throughput sequencing to study the role of lncRNA in leukemia cell lines on a large scale.

Leukemia Cells Resistant to Glutamine Deprivation Express Glutamine Synthetase Protein

Objective:Low glutamine levels have been shown in tumor environments for several cancer subtypes. Therefore, it has been suggested that cancer cells rewire their metabolism to adopt low nutrient levels for survival and proliferation. Although glutamine is a non-essential amino acid and can be synthesized de novo, many cancer cells including malignant hematopoietic cells have been indicated to be addicted to glutamine. This study aimed to investigate the proliferation of leukemia cell lines in glutamine-deprived conditions.Materials and Methods:Cell proliferation of K562, NB-4, and HL-60 cells was determined by calculating cell numbers in normal vs. low glutamine media. Changes in mRNA expressions were investigated using qRT-PCR. The glutamine synthetase (GS)-encoding GLUL gene was knocked out (KO) in HL-60 cells using the CRISPR/Cas9 method and protein expression was evaluated with immunoblotting.Results:The proliferation of all cell lines was decreased in glutamine-deprived medium. GS protein expression was increased in glutamine-limited medium although the mRNA level did not change. Increased protein expression was confirmed with inhibition of new protein synthesis by treating cells with cycloheximide. To further investigate the role of GS protein, the GS-encoding GLUL gene was KO in HL-60 cells using the CRISPR/Cas9 method. GS KO cells proliferated less compared to control cells in glutamine-limited medium.Conclusion:Our results indicate that upregulated GS protein expression is responsible for glutamine addiction of leukemia cell lines. Exploiting the genetic and metabolic mechanisms responsible for GS protein expression could lead to the identification of new anti-cancer drug targets.

Development of an MDM2 Degrader for Treatment of Acute Leukemias

IntroductionAcute myeloid leukemias (AMLs) are characterized by suppressed cell death pathways which promote leukemic blast survival. TP53 acts as a tumor suppressor gene in AML and is found mutated or deleted in 10-15% of patients. In a majority of cases though, TP53 is wild-type. Other mechanisms including MDM2 over-expression lead to reduced TP53 activity. MDM2 acts as a negative regulator by direct binding of TP53 and mediating TP53 degradation through ubiquitination. MDM2 itself is a transcriptional target of TP53 as a negative feedback mechanism limiting the function of TP53. Small molecule inhibition of MDM2 , blocking its ability to bind TP53, can activate TP53 and trigger cell cycle arrest and apoptosis through increased transcription of TP53 target genes. Increased MDM2 expression has been observed in hematologic malignancies including AML, providing rationale for clinical trials with MDM2 inhibitors. These agents such as RG7388 and AMG232 have shown efficacy as monotherapy and in combination. However, these agents have also exhibited toxicity and have yet to demonstrate sufficient benefit for their approval. To create more effective agents against MDM2, we have developed an MDM2 degrader XY-27 that functions as a proteolysis-targeting chimera (PROTAC). Based on relatively higher expression in AML compared to other cancer types, we selected VHL as the E3 ubiquitin ligase target for XY-27 , as this may improve specificity and potency in AML.ResultsThe PROTAC degrader XY-27 concurrently binds MDM2 and VHL, and by bringing these targets in proximity, VHL can then ubiquitinate MDM2, leading to its degradation by the proteasome. XY-27 can mediate degradation of MDM2 in a concentration dependent manner in the U937 leukemia cell line (Fig 1a). MDM2 degradation with XY-27 is blocked by proteasome inhibition and competitive binding of the VHL ligand. A control compound, which only differs in that it cannot bind to VHL, lacks degrader activity. Although MDM2 is itself an E3 ligase, VHL expression is not appreciably changed with XY-27 (Fig 1a).Treatment with XY-27 leads to apoptosis and decreased proliferation of leukemia cell lines in a TP53 dependent manner. Inhibition of MDM2 leads to up-regulation of TP53 and in TP53 wild-type cells, downstream targets CDKN1A (p21) and PUMA. MDM2 is also up-regulated through a feedback mechanism. XY-27 demonstrated greater potency than the MDM2-binding inhibitor AMG232 in the MOLM13 and MV4-11 leukemia cell lines (Fig 1b). Treatment with XY-27 led to higher levels of TP53 and p21 protein than with AMG232. CRISPR-mediated knock-out of VHL leads to reduced XY-27 potency. XY-27 also shows efficacy when combined with other chemotherapeutic agents such as azacytidine and cytarabine. In a long-term co-culture model with an OP9 feeder layer, XY-27 was capable of inducing apoptosis in primary patient AML samples (Fig 1c).ConclusionWe describe a new MDM2 PROTAC, XY-27 that demonstrates TP53 dependent activity against leukemia cells. It also demonstrates increase potency compared to an MDM2 binding inhibitor. Utilization of the PROTAC system has potential advantages through selection of the VHL E3 ubiquitin ligase. Because of negative feedback mechanisms involving TP53 and MDM2, direct binding inhibitors of MDM2 may be limited in activity through continued accumulation of MDM2. PROTAC degraders have catalytic activity and may overcome this inhibition by continued degradation of the target MDM2, and thus achieve greater TP53 activity.Figure 1. Activity of the MDM2-PROTAC XY-27 in leukemia. (a) Western blot from treatment of U937 leukemia cells with XY-27 for 24 hrs, at various concentrations (5 nM to 1 μM), resulting in the degradation of MDM2. (b) Dose response curves from treatment of MOLM13 and MV4-11 cell lines with XY-27 (blue) and AMG232 (red) for 48 hrs, demonstrating greater potency of XY-27. (c) Induction of apoptosis in primary AML cells treated with XY-27 at 1μM using a co-culture system for 3 days. *p<.05 [Display omitted] DisclosuresHoffman: Protagonist Therapeutics, Inc.: Consultancy; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding; Novartis: Other: Data Safety Monitoring Board, Research Funding; Kartos Therapeutics, Inc.: Research Funding.

Targeted CRM1-inhibition perturbs leukemogenic NUP214 fusion proteins and exerts anti-cancer effects in leukemia cell lines with NUP214 rearrangements

Chromosomal translocations fusing the locus of nucleoporin NUP214 each with the proto-oncogenes SET and DEK are recurrent in, largely intractable, acute leukemias. The molecular basis underlying the pathogenesis of SET-NUP214 and DEK-NUP214 are still poorly understood, but both chimeras inhibit protein nuclear export mediated by the β-karyopherin CRM1. In this report, we show that SET-NUP214 and DEK-NUP214 both disturb the localization of proteins essential for nucleocytoplasmic transport, in particular for CRM1-mediated protein export. Endogenous and exogenous SET-NUP214 and DEK-NUP214 form nuclear bodies. These nuclear bodies disperse upon targeted inhibition of CRM1 and the two fusion proteins re-localize throughout the nucleoplasm. Moreover, SET-NUP214 and DEK-NUP214 nuclear bodies reestablish shortly after removal of CRM1 inhibitors. Likewise, cell viability, metabolism, and proliferation of leukemia cell lines harboring SET-NUP214 and DEK-NUP214 are compromised by CRM1 inhibition, which is even sustained after clearance from CRM1 antagonists. Our results indicate CRM1 as a possible therapeutic target in NUP214-related leukemia. This is especially important, since no specific or targeted treatment options for NUP214 driven leukemia are available yet.

Exogenous Expression of WNT7A in Leukemia-Derived Cell Lines Induces Resistance to Chemotherapeutic Agents.

Dysregulations of the WNT pathway are implicated in the malignant transformation of different types of neoplasia. WNT7A is expressed in normal peripheral lymphocytes, but is decreased in the tumoral counterpart. Furthermore, the treatment of leukemic cells with recombinant WNT7A decreases proliferation, suggesting its possible use as a therapeutic biomolecule. This study aimed to evaluate the concomitant action of WNT7A and different chemotherapeutic agents over proliferation and cell death of leukemia/ lymphoma derived cell lines. Ectopic expression of WNT7A was induced in CEM and BJAB cell lines by using a lentiviral system. RNA expression was analyzed by microarrays and qPCR, and protein expression was determined by Western Blot. Cell proliferation was measured by cell counting, metabolic activity by WST-1 assay, cell death and DNA content by flow cytometry. WNT7A ectopic expression was shown to decrease cell proliferation, but the apoptosis rate of leukemic cells was not altered. Moreover, these cells acquired resistance to doxorubicin, vincristine and MG-132. Cell cycle analysis reveals a decrease in G1 and an increase in S and G2 phases with a further upregulation of senescence- associated genes. Microarray analysis reveals that most gene expression changes were related to cancer and metabolic associated pathways. All those changes appear to be independent of the WNT canonical pathway regulation. WNT7A negatively regulates cell proliferation in leukemic cell lines and promotes resistance to chemotherapeutic agents by inducing a senescence-like phenotype independently of the WNT canonical pathway.

Inhibition of PI3K/mTOR Pathways with GDC-0980 in Pediatric Leukemia: Impact on Abnormal FLT-3 Activity and Cooperation with Intracellular Signaling Targets.

GDC-0980 is a selective small molecule inhibitor of class I PI3K and mTOR pathway with a potent anti-proliferative activity. We set out to evaluate the efficacy of GDC-0980, in pre-clinical studies, against pediatric leukemia cells. The anti-neoplastic activity of GDC-0980 was evaluated in vitro using five different pediatric leukemia cells. Our data show that GDC-0980 significantly inhibited the proliferation of leukemia cell lines, KOPN8 (IC50, 532 nM), SEM (IC50,720 nM), MOLM-13 (IC50,346 nM), MV4;11 (IC50,199 nM), and TIB-202 (IC50, 848 nM), compared to normal control cells (1.23 µM). This antiproliferative activity was associated with activation of cellular apoptotic mechanism characterized by a decrease in Bcl-2 protein phosphorylation and enhanced PARP cleavage. Western blot analyses of GDC-0980 treated cells also showed decreased phosphorylation levels of mTOR, Akt and S6, but not ERK1/2. Notably, FLT3 phosphorylation was decreased in Molm-13 and MV4;11 cells following the application of GDC-0980. We further examined cellular viability of GDC-0980-treated primary leukemia cells isolated from pediatric leukemia patients. This study revealed a potential therapeutic effect of GDC-0980 on two ALL patients (IC50's, 1.23 and 0.625 µM, respectively). Drug combination analyses of GDC-0980 demonstrated a synergistic activity with the MEK inhibitor Cobimetinib (MV4-11; 11, CI, 0.25, SEM, CI, 0.32, and TIB-202, CI, 0.55) and the targeted FLT3 inhibitor, Crenolanib (MV4-11; 11, CI, 0.25, SEM, CI, 0.7, and TIB-202, CI, 0.42). These findings provide initial proof-of-concept data and rationale for further investigation of GDC-0980 in selected subgroups of pediatric leukemia patients.

Effects of a Traditinal Chinese Medicine Compound Regimen on Differentiation and Proliferation of Acute Myeloid Leukemia Cell Lines

To study the effect of traditional chinese medicine (TCM) compound on myeloid leukemia cells and to explore its anti-leukemic mechanism. Myeloid leukemia cell lines were cultured in vitro and treated with TCM compound. The proliferation of the leukemia cells was measured by CCK8 method. The differentiation of the leukemia cells was evaluated by using Wright＇s staining method and by light microscopy, and the expression of differentiation-related surface antigens such as CD11B was measured and by flow cytometry, the apoptosis of the leukemia cells was detected by flow cytometry with using Annexin V staining. Compared with untreated 4 leukemia cell lines HL-60, MOLM-13, MV4-11, AML-M5, the proliferations of 4 leukemia cells treated with different concentrations of TCM compound decreased (P<0.05), and their proliferation inhibition were in a dose-dependent manner (r=0.9236; r=0.7488; r=0.8889; r=0.8119); compared with HL-60 and AML-M5 leukemia cells, the drug-treated 2 leukemia cells displayed obvious differentiated changes; compared with untreated HL-60 leukemia cell line, the expression of surface antigen CD11B increased by 85%±7.13% in HL-60 cells treated IC50 concentration of drug; compared with untreated AML-M5 leukemia cell line, the apoptotic rate of AML-M5 treated with 1.5 and 2 μl doses of TCM compound increased. (P<0.05). The traditional chinese medicine compound may inhibit the proliferation of leukemia cell lines mainly by inducing leukemia cell differentiation and apoptosis.

3-Aryl/Heteroaryl-5-amino-1-(3′,4′,5′-trimethoxybenzoyl)-1,2,4-triazoles as antimicrotubule agents. Design, synthesis, antiproliferative activity and inhibition of tubulin polymerization

Many natural and synthetic substances are known to interfere with the dynamic assembly of tubulin, preventing the formation of microtubules. In our search for potent and selective antitumor agents, a novel series of 1-(3′,4′,5′-trimethoxybenzoyl)-5-amino-1,2,4-triazoles were synthesized. The compounds had different heterocycles, including thiophene, furan or the three isomeric pyridines, and they possessed a phenyl ring bearing electron-releasing or electron-withdrawing substituents at the 3-position of the 5-amino-1,2,4-triazole system. Most of the twenty-two tested compounds showed moderate to potent antiproliferative activities against a panel of solid tumor and leukemic cell lines, with four (5j, 5k, 5o and 5p) showing strong antiproliferative activity (IC50 < 1 μM) against selected cancer cells. Among them, several molecules preferentially inhibited the proliferation of leukemic cell lines, showing IC50 values 2-100-fold lower for Jurkat and RS4;11 cells than those for the three lines derived from solid tumors (HeLa, HT-29 and MCF-7 cells). Compound 5k strongly inhibited tubulin assembly, with an IC50 value of 0.66 μM, half that obtained in simultaneous experiments with CA-4 (IC50 = 1.3 μM).

Down regulation of G protein-coupled receptor 137 expression inhibits proliferation and promotes apoptosis in leukemia cells

BackgroundG protein-coupled receptors (GPR) are involved in a wide range of physiological processes, some of which, however, can be hijacked by tumor cells. Over-expression of G protein-coupled receptors 137 (GPR137) are associated with the growth of tumor cells, but under-expression of GPR137 has shown to inhibit cell proliferation in several different types of cancers. Currently, the role of GPR137 in leukemia is still unclear. In this study, the effect of under-expression of GPR137 on inhibiting the proliferation of leukemia cells is explored, to identify a novel target for leukemia treatment.Materials and methodsIn this study, lentivirus-mediated RNA interference (RNAi) was employed to investigate the role of GPR137 in two leukemia cell lines K562 and HL60. The gene expression of GPR137 was analyzed by RT-PCR and its protein expression was determined by Western blot. Flow cytometry and Annexin V/7-AAD Apoptosis Detection Kit was used respectively in cell cycle and apoptosis analysis. The protein expression of CyclinD1, CDK4, BCL-2 and caspase-3 were also determined.ResultsThere was high level of constitutive expression of GPR137 in leukemia cancer cell lines K562 and HL60. Lentivirus-mediated RNAi could significantly down-regulate gene and protein expression of GPR137 in both cell lines. Down regulation of GPR137 was associated with the reduction in proliferation rate and colony forming capacity. In addition, down regulation of GPR137 arrested cells in the G0/G1 phase of cell cycle and induced apoptosis in both leukemia cell lines K562 and HL60.ConclusionsThe expression of GPR137 is associated with the proliferation of leukemia cell lines. Down regulation of GPR137 could inhibit proliferation and promote apoptosis in leukemia cells, which makes it a promising bio-marker and therapeutic target to treat patients with leukemia.

N‐methyl‐d‐aspartate receptor mediated calcium influx supports in vitro differentiation of normal mouse megakaryocytes but proliferation of leukemic cell lines

Unlabelled Box BackgroundN‐methyl‐d‐aspartate receptors (NMDARs) contribute calcium influx in megakaryocytic cells but their roles remain unclear; both pro‐ and anti‐differentiating effects have been shown in different contexts. ObjectivesThe aim of this study was to clarify NMDAR contribution to megakaryocytic differentiation in both normal and leukemic cells. MethodsMeg‐01, Set‐2, and K‐562 leukemic cell lines were differentiated using phorbol‐12‐myristate‐13‐acetate (PMA, 10 nmol L−1) or valproic acid (VPA, 500 μmol L−1). Normal megakaryocytes were grown from mouse marrow‐derived hematopoietic progenitors (lineage‐negative and CD41a‐enriched) in the presence of thrombopoietin (30‐40 nmol L−1). Marrow explants were used to monitor proplatelet formation in the native bone marrow milieu. In all culture systems, NMDARs were inhibited using memantine and MK‐801 (100 μmol L−1); their effects compared against appropriate controls. ResultsThe most striking observation from our studies was that NMDAR antagonists markedly inhibited proplatelet formation in all primary cultures employed. Proplatelets were either absent (in the presence of memantine) or short, broad and intertwined (with MK‐801). Earlier steps of megakaryocytic differentiation (acquisition of CD41a and nuclear ploidy) were maintained, albeit reduced. In contrast, in leukemic Meg‐01 cells, NMDAR antagonists inhibited differentiation in the presence of PMA and VPA but induced differentiation when applied by themselves. ConclusionsNMDAR‐mediated calcium influx is required for normal megakaryocytic differentiation, in particular proplatelet formation. However, in leukemic cells, the main NMDAR role is to inhibit differentiation, suggesting diversion of NMDAR activity to support leukemia growth. Further elucidation of the NMDAR and calcium pathways in megakaryocytic cells may suggest novel ways to modulate abnormal megakaryopoiesis.

Loperamide, an antidiarrheal agent, induces apoptosis and DNA damage in leukemia cells.

Loperamide, an antidiarrheal agent, is frequently used to treat patients with leukemia with symptoms of diarrhea during treatment. However, the effect of loperamide on leukemia cells is unknown. The MTT assay was used to explore the cytotoxic effect of loperamide on leukemia cells. Morphological analysis and flow cytometry were performed to determine the level of apoptosis in leukemia cells following loperamide treatment. Western blotting was conducted to test the activation of the apoptotic pathway. The comet assay was used to determine the DNA damage induced by loperamide. Loperamide potently inhibited the proliferation of leukemia cell lines and primary leukemia cells from 9 patients with acute myeloid leukemia (AML) and 6 patients with acute lymphocytic leukemia (ALL) in a dose-dependent manner. Loperamide increased the expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, decreased the expression of myeloid cell lekeumia-1 and induced the apoptosis of leukemia cells. In addition, treatment with 20 µM loperamide increased the expression level of the protein rH2ax and promoted the formation of long DNA comet tails, thus triggering DNA damage in leukemia cells. Finally, DNA damage was confirmed by the activation of the ataxia telangiectasia mutated serine/threonine kinase (ATM)-checkpoint kinase 2 (Chk2) signaling pathway. The phosphorylation level of ATM (Ser1981) and Chk2 (Thr68) was activated and upregulated following DNA damage triggered by loperamide. Loperamide was demonstrated to perform an inhibitory role in the growth of leukemia cell lines and primary leukemia cells. Of note, apoptosis and DNA damage were induced following loperamide treatment in leukemia cell lines and primary leukemia cells.

Anti-invasive and antiproliferative effects of Pleurotus ostreatus extract on acute leukemia cell lines.

Currently, mushrooms have been used in traditional and folk medicines for their therapeutic activities, such as antibiotic, antitumor, anti-inflammatory, anticancer, antileukemic and immunomodulatory actions. This investigation evaluates the anti-invasive, antiproliferative and cytotoxic effects of Pleurotus ostreatus (Pleurotaceae) on leukemia cell lines. The proliferation of KG-1 cells was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with gradient dilutions of P. ostreatus extract. Then, the minimum inhibitory concentration (MIC) of the extract was determined. Moreover, the proliferation of Jurkat cells and bone marrow mesenchymal stem cells (BMSCs), a cancerous cell line and normal body cells, respectively, was considered. The apoptotic morphology of treated KG-1 cells was evaluated with Giemsa staining. The invasion and migration of cells were evaluated using transwell invasion assay. Thereafter, the rates of apoptosis and necrosis were measured by using flow cytometry, and BAX and MMP-9 gene expression were evaluated using quantitative reverse transcription-polymerase chain reaction as apoptotic and metastatic genes, respectively. The MIC of the extract was determined to be 1 mg/mL after 48 h. According to the results, the extract decreased the proliferation of leukemia cell lines (KG-1 and Jurkat cells) but had no antiproliferative effects on BMSCs. Moreover, KG-1 cell migration and MMP-9 gene expression decreased after the treatment, and the rate of apoptosis and BAX gene expression increased significantly. According to the efficient therapeutic properties of P. ostreatus on leukemia cell lines, this mushroom could be introduced as a natural medicine to cure leukemic patients who suffer from the harmful side effects and enormous costs of chemotherapy.

Leukemia-Derived Exosomes Induce Paracrine and Autocrine Cell Proliferation in Pediatric ALL

Introduction: Liquid biopsies are minimal invasive and changing our understanding of tumor cell biology through the exploration of exosomes research in adult oncology. The clinical application of liquid biopsies to study exosomes as disease biomarkers with correlation to disease status is especially appealing in pediatrics. However in childhood cancer, the first exosome studies only begin to emerge in this exciting new field. Exosomes are microvesicles (30-100 nm) produced by normal as well as malignant cells in most biological fluids. Exosomes represent the fingerprint of the parental tumor and are loaded with unique biomarkers such as proteins, RNA, DNA and non-coding RNA, which may regulate tumor growth. One of the unique features of exosomes of interest to oncology research is the horizontal cargo transfer into target cells changing their biological properties. Our data present the first study to investigate the role of exosomes in acute lymphoid leukemia (ALL) in pediatric patients.Objectives:1. Demonstrate transfer of ALL-exosomes into target cells with induction of cell proliferation2. Confirm that leukemic exosome-induced paracrine and autocrine cell proliferation of leukemic cell lines is regulated by expression of proliferative and pro-survival genes with suppression of pro-apoptotic genes.Methods: Exosomes were isolated from serum of healthy donors (HD) and pediatric ALL (P-ALL) patients by ultracentrifugation. Exosomes were also isolated from conditioned medium (CM) of SUP-B15 (B-ALL), JM1 (B-ALL), and CL-01 (normal B cells) human cell lines. Proliferation assays were designed by exposing all three cell lines to different sources of leukemia-derived exosomes in a paracrine or autocrine fashion. After 24hr incubation, induction of cell proliferation was assessed by cell counting using trypan blue under the microscope and confirmed by colorimetric assay and gene expression analysis. Total RNA was extracted from cultured SUP-B15, JM1, or CL-01 cells and 2-5 µg of total RNA was used for cDNA synthesis. Primers sequences, probe number and gene accession numbers were identified from the universal probe library (UPL) of Roche Applied Science (Indianapolis, IN, USA). Each sample was assayed in duplicates. Proliferation was evaluated at the gene expression level for the following genes: Ki-67, PCNA (proliferation), MCL1, BCL2 (pro-survival) and BAD, BAX (pro-apoptotic). Fold change was calculated by comparing controls (naive) vs. exosome-induced cell lines both in autocrine and paracrine fashion. Data were analyzed with RQ manager version 1.2.1 (Applied Biosystems, Foster City, CA). The data were expressed as relative mRNA expression with reference to control samples and normalized to an endogenous reference gene (GAPDH).Results: Exosomes were characterized by NanoSight Tracking Analysis (NTA-Malvern) and western blot. Exosomal uptake and internalization into the cells was visualized by exosome labeling with a PKH67 fluorescent dye. We elucidated that CM-derived exosomes from SUP-B15 and JM1 cell lines induce cell proliferation in SUP-B15, JM1 (autocrine and paracrine for both cell lines) and CL-01 cells (paracrine) (control vs. exosomes, p<0.01). Moreover, P-ALL serum-derived exosomes promote paracrine cell proliferation in SUP-B15, JM1 and CL-01 cell lines compared to HD-derived exosomes (HD vs P-ALL, p<0.0001). At a molecular level, we found that exosomes from JM1 and SUP-B15 cells enhance expression of proliferation genes (PCNA, Ki-67) and pro-survival genes (MCL-1, BCL2), and suppress pro-apoptotic genes (BAD, BAX) in JM1 cells.Conclusions: Our data suggest that human ALL-derived exosomes induce cell proliferation in both paracrine and autocrine fashion of leukemic and non-leukemic B cell lines. It appears that exosomes regulates this phenomena in a highly orchestrated way, by the induction of proliferative genes and pro-survival genes, with suppression of pro-apoptotic genes. DisclosuresNo relevant conflicts of interest to declare.

PLK-1 Expression is Associated with Histopathological Response to Neoadjuvant Therapy of Hepatic Metastasis of Colorectal Carcinoma

Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and overexpressed in multiple human cancers, including leukemia and also many solid tumors. PLK1 knockdown has been shown to block proliferation of leukemic cell lines and the clonogenic potential of tumor cells grown from patients with cancer. PLK1 inhibition is a promising strategy for the treatment of some tumors. We aim to analyze expression of PLK1 in metastatic colorectal carcinoma. Retrospective analysis of colorectal carcinomas with hepatic metastasis during follow-up receiving neoadjuvant chemotherapy (NAC), based on oxaliplatin. Immunohistochemistry for PLK-1 in paraffin-embedded tissue from the primary and also from the metastasis. 50 patients. 32% showed good histopathological response. 43% of the primaries were positive for PLK1, as opposed to 23.5% of the metastasis. Expression of PLK1 was significantly reduced in metastasis compared with the primaries (p = 0.05), what could be due to therapy or to a phenotypic change of the metastatic nodule. Analysis of the prognostic influence of PLK1 expression showed significant association between PLK1 expression in metastasis and lower overall survival (p = 0.000). We have also found a significant association between PLK1 expression and histopathological response (p = 0.02). All the tumors with high expression of PLK1 showed minor response (11/11). This study shows the association between survival and poor histopathological response to therapy and high expression of PLK1 in metastasis. Our results could open a new therapeutic approach through the inhibition of PLK1.

Statin-mediated inhibition of cholesterol synthesis induces cytoprotective autophagy in human leukemic cells

Statins exhibit anti-leukemic properties due to suppression of the mevalonate pathway by the inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase, and subsequent depletion of cholesterol, farnesylpyrophosphate, and geranylgeranylpyrophosphate. We investigated the role of autophagy, a controlled intracellular self-digestion, in the anti-leukemic action of statins. Treatment with low concentrations (≤6µM) of statins, cholesterol depletion, and specific inhibition of cholesterol synthesis and protein farnesylation or geranylgeranylation, all inhibited proliferation of leukemic cell lines and primary leukemic cells without inducing overt cell death. Statins and agents that selectively reduce intracellular cholesterol levels, but not the inhibition of protein farnesylation or geranylgeranylation, induced autophagy in leukemic cells. The observed autophagic response was associated with the reduction of phosphorylated Akt levels in the lipid rafts, accompanied by a decrease in the activation of the main autophagy suppressor mammalian target of rapamycin (mTOR) and its substrate ribosomal p70S6 kinase (p70S6K). No significant autophagy induction and downregulation of mTOR/p70S6K activation were observed in normal leukocytes. Autophagy suppression by bafilomycin A1 or RNA interference-mediated knockdown of beclin-1 and microtubule-associated protein 1 light chain 3B induced apoptotic death in statin-treated leukemic cells, an effect attenuated by the addition of mevalonate or squalene, but not farnesylpyrophosphate or geranylgeranylpyrophosphate. Therefore, while the inhibition of cholesterol synthesis, protein farnesylation, and geranylgeranylation all contributed to anti-leukemic effects of statins, the inhibition of cholesterol synthesis was solely responsible for the induction of cytoprotective autophagy. These data indicate that combined treatment with statins and autophagy inhibitors might be potentially useful in anti-leukemic therapy.

Inhibition of the Na+/H+ antiporter induces cell death in TF-1 erythroleukemia cells stimulated by the stem cell factor

Leukemia cells produce acidic metabolites due to their high metabolic condition. An alkaline pHi (intracellular pH) shift, caused by activation of the Na+/H+ exchange, is an important event in the mechanism of growth factor activity. However, the role of the Na(+)/H(+) exchanger in the survival of erythroleukemia TF-1 cells has not yet been studied in detail. The aim of this study was to identify the effects of 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a highly specific blocker of the Na(+)/H(+) exchanger, on the survival of SCF-dependent TF-1 cells. The effects of EIPA on survival and mitochondrial membrane potential were studied when exposing wild type TF-1 cells and TF-1 cells expressing bcl-2 to EIPA for 48h. Ectopic expression of the bcl-2 gene maintained a mildly alkaline pH and prevented the simultaneous appearance of apoptosis and autophagy (typically displayed by TF-1 cells) in the presence of EIPA. Consistent with Stem Cell Factor (SCF) function, we found that this molecule rescued TF-1 cells during autophagy but not apoptosis, allowing these cells to subsequently respond to GM-CSF. Serum deprivation or SCF withdrawal induced cell death at 36h in TF-1 and TF-1 neo cells, whereas TF-1/bcl-2 cells tended to undergo apoptosis and show acidic vacuoles after 96h, pointing to a transient anti-apoptotic effect. The present study shows the suppressive effect of EIPA on the proliferation of leukemia cell line stimulated with SCF, apparently by decreasing the mitochondria membrane potential and averting alkalinization. Through the constitutive expression of bcl-2, TF-1 cells were survival factor independent. Proliferation in these cells was not affected by EIPA at the concentrations used against parental TF-1 cells, indicating that the inhibitory effect in SCF-stimulated cells can be attributed to specific blocking of the Na(+)/H(+) exchanger.

Effects of silver nanoparticles and gold nanoparticles on IL-2, IL-6, and TNF-α production via MAPK pathway in leukemic cell lines.

Silver nanopaticles (AgNPs) and gold nanoparticles (AuNPs) have various applications in medical healthcare and various biological properties such as anti-inflammation, anti-cancer, and anti-angiogenesis. In this study, we investigated the effect of AgNPs and AuNPs on cytokine production via the mitogen-activated protein kinase pathway in leukemic cell lines (T-lymphocytic Jurkat and monocytic U937 cells). We found that both AgNPs and AuNPs inhibited cell proliferation of leukemic cell lines. AgNPs inhibited TNF-α, while AuNPs inhibited interleukin-2 production in Jurkat cells, in which inhibition of cytokines is involved in the extracellular-signal regulated protein kinase but not the c-Jun N-terminal kinase pathway. In U937 cells, AuNPs inhibited interleukin-6 but stimulated TNF-α production in a concentration-dependent manner through the c-Jun N-terminal kinase but not the extracellular-signal regulated protein kinase pathway. Our study showed that each leukemic cell line treated with nanoparticles exhibited a distinct signaling pathway response to inhibit or stimulate cytokine production, leading to anti-cell proliferation. The effects of AgNPs and AuNPs on leukemic cell lines may have a significant impact on leukemia treatment in the future.