Sensitizes Acute Lymphoblastic Leukemia Cells Research Articles

C-Myc Inhibition Induces an Additive Effect with Cyclophosphamide in Acute Lymphoblastic Leukemia

Background: Cellular-myelocytomatosis (c-Myc), an oncoprotein and a transcription factor, is involved in several essential cellular processes. The c-Myc expression level is highly regulated in normal cells. It has been proved that c-Myc expression is deregulated in malignant cells due to rearrangements and mutations. The overexpression of this molecule is also reported to be present in acute lymphoblastic leukemia (ALL) as well, which is correlated with an unfavorable response to treatment, poor prognosis, and decreased overall survival. The upregulation of c-Myc results in increased proliferation, cell growth, and survival of ALL cells. Hence, making it an ideal target for leukemia treatment. This study evaluates the effect of c-Myc silencing combined with cyclophosphamide treatment, an FDA-approved chemotherapeutic. Methods: Peripheral blood and bone marrow samples (mononuclear cells) were derived from eleven ALL patients. To silence c-Myc, small interfering-RNA (siRNA)-lipofectamine was used. The efficacy of gene silencing was assessed by the qRT-PCR test. Next, the effect of c-Myc silencing combined with cyclophosphamide treatment in ALL primary cells was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) test. Results: ALL cells were successfully transfected with c-Myc-siRNA. Also, treating cells with cyclophosphamide exerted a slight fall in the c-Myc mRNA level. The MTT test revealed that following the inhibition of c-Myc by siRNA, the viability of primary ALL cells decreased in response to cyclophosphamide treatment. Also, it was discovered that silencing c-Myc with siRNA combined with cyclophosphamide treatment significantly inhibits the growth of primary ALL cells compared to cyclophosphamide monotherapy. Conclusion: c-Myc possesses high potential in the treatment of several cancers. Our findings add ALL to this category as well. Silencing c-Myc sensitizes ALL cells to cyclophosphamide treatment and can help with the better treatment of the afflicted individuals.

Ferroptosis Patterns Predicting Prognosis and Drug Resistance in Acute Lymphoblastic Leukemia

Background: Most acute lymphoblastic leukemia (ALL) patients are treated with chemotherapy as primary care. Although treatment response is usually positive, resistance and relapse often occur through unclear mechanisms. Ferroptosis is an iron-dependent form of regulating cell death associated with cancer. The characteristics of ferroptosis in ALL are still uncertain. This study aimed to explore the effect of ferroptosis on drug resistance and prognosis, and the potential of ferroptosis-based treatment in ALL. Methods: We comprehensively evaluated the ferroptosis levels of 480 ALLs from the TARGET and GEO database, and 99 ALLs from our clinic based on 487 ferroptosis-related specific genes (FRGs) downloaded from FerrDb database. The ferroptosis score (FS) was constructed to quantify the ferroptosis levels of individuals using principal component analysis algorithms. Next, the prognostic value and therapeutic sensitivities were evaluated using multiple methods. We then assessed the transcription profile of 99 ALLs in our clinic according to the minimal residual diseased (MRD) level, and the integrated metabolic analysis and RNA sequencing in drug-resistant and sensitive ALL cell lines. The biological function of solute carrier family 7 member 11 (SLC7A11), one of FRGs, on cell growth, lipid peroxidation, and drug sensitivity were further examined in ALL cell lines Nalm-6, Jurkat, and their corresponding drug-resistant cells Nalm-6/ADR and Jurkat/ADR. Results: At first, we identified that FRGs have prevalent genetic alteration in ALL, most for missense mutation of driver genes. In addition, the heterogeneous expression of the FRGs in normal and ALL samples indicated the involvement of FRGs expression in ALL development. Subsequently, we identified two distinct ferroptosis phenotypes in ALL with significantly different prognoses. We then developed a risk model termed FS to evaluate ferroptosis levels of ALL patients based on differential expression genes between the two ferroptosis phenotypes. Compared with the low-FS group, the high-FS group was characterized by higher estimated IC50 in the many chemotherapy drugs with an unfavorable prognosis, which was validated by our clinical data. Furtherly, SLC7A11 was identified as a key regulator of ferroptosis-related drug resistance in ALL. Clinically, SLC7A11 was selected out with the highest log |FC| in the MRDHigh(>1%) group in comparison with MRDlow(＜0.01%) group. The abundance of SLC7A11 was strongly correlated with relapsed/refractory disease. Overexpression of SLC7A11 and reduction of ferroptosis were observed in the drug-resistant cells. The SLC7A11-specific classic ferroptosis inducer erastin and shRNA both decreased cellular glutamine uptake, increased cellular lipid peroxidation, enhanced the sensitivity of chemotherapeutic with synergism, and led to deceleration of cell proliferation via glutamate metabolism in the oxidative phosphorylation pathway. Conclusions: Ferroptosis score could be a prognostic indicator for ALL progression and chemotherapy resistance. SLC7A11 functioned as an extracellular glutamine transporter, promoting ALL resistance through reprogramming glutamine metabolism of ALL, while rendering ALL cells insensitive to chemotherapy. Ferroptosis-based therapies employing agents to target SLC7A11 offered substantial therapeutic improvements over conventional chemotherapy for ALL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Amino Acid Stress Response Genes Promote L-Asparaginase Resistance in Pediatric Acute Lymphoblastic Leukemia

▪Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcome. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood, beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP resistant and sensitive ALL cell lines (n = 7) as well as primary leukemia samples from newly diagnosed patients. We identified 2219 response genes (absolute log 2FC > 1.5, FDR p-value <0.05) with ~16.5% being reproduced in more than one cell line. Defining target genes of the amino acid stress response related transcription factor ATF4 in ALL cell lines using ChIP-seq revealed 25% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor. A total of 17,117 significantly differentially bound ATF4 sites were identified (FDR p-value <0.01) and 97.8% of these sites displayed an increase in ATF4 binding following LASP treatment. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells from 212 newly diagnosed children enrolled on St. Jude Total Therapy 16. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlights that many response genes may not differ in their basal expression in drug resistant leukemia cells. DisclosuresStock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. Mullighan: Amgen: Current equity holder in publicly-traded company; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Pfizer: Research Funding. Pui: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Data Monitoring Committee. Evans: Princess Máxima Center for Pediatric Oncology, Scientific Advisory Board, Chair: Membership on an entity's Board of Directors or advisory committees; BioSkryb, Inc.: Membership on an entity's Board of Directors or advisory committees; St. Jude Children's Research Hospital, Emeritus Member (began Jan 2021): Ended employment in the past 24 months.

CD81 knockout promotes chemosensitivity and disrupts in vivo homing and engraftment in acute lymphoblastic leukemia

AbstractRelapse remains a major obstacle to achieving 100% overall survival rate in pediatric hematologic malignancies like acute lymphoblastic leukemia (ALL). Relapse often results from the development of chemoresistance. One of the mechanisms of chemoresistance involves ALL cell interactions with the bone marrow (BM) microenvironment, providing a sanctuary. This phenomenon is known as BM microenvironment-induced chemoprotection. Members of the transmembrane 4 superfamily (tetraspanins; TSPANs) are known to mediate microenvironmental interactions and have been extensively studied in solid tumors. Although the TSPAN family member CD81 is a minimal residual disease marker, its biological role in ALL is not well characterized. We show for the first time that CD81 knockout induces chemosensitivity, reduces cellular adhesion, and disrupts in vivo BM homing and engraftment in B-ALL. This chemosensitization is mediated through control of Bruton tyrosine kinase signaling and induction of p53-mediated cell death. We then show how CD81-related signaling can be disrupted by treatment with the epigenetic drug combination of DNA hypomethylating agent azacitidine (aza) and histone deacetylase inhibitor panobinostat (pano), which we previously used to sensitize ALL cells to chemotherapy under conditions that promote BM microenvironment-induced chemoprotection. Aza/pano-mediated modulation of CD81 surface expression is involved in decreasing BM load by promoting ALL cell mobilization from BM to peripheral blood and increasing response to chemotherapy in disseminated patient-derived xenograft models. This study identifies the novel role of CD81 in BM microenvironment-induced chemoprotection and delineates the mechanism by which aza/pano successfully sensitizes ALL cells via modulation of CD81.

Epigenetic drug combination overcomes osteoblast-induced chemoprotection in pediatric acute lymphoid leukemia

Although there has been much progress in the treatment of acute lymphoblastic leukemia (ALL), decreased sensitivity to chemotherapy remains a significant issue. Recent studies have shown how interactions with the bone marrow microenvironment can protect ALL cells from chemotherapy and allow for the persistence of the disease. Epigenetic drugs have been used for the treatment of ALL, but there are no reports on whether these drugs can overcome bone marrow-induced chemoprotection. Our study investigates the ability of the DNA methyltransferase inhibitor azacitidine and the histone deacetylase inhibitor panobinostat to overcome chemoprotective effects mediated by osteoblasts. We show that the combination of azacitidine and panobinostat has a synergistic killing effect and that this combination is more effective than cytarabine in inducing ALL cell death in co-culture with osteoblasts. We also show that this combination can be used to sensitize ALL cells to chemotherapeutics in the presence of osteoblasts. Finally, we demonstrate that these effects can be replicated ex vivo in a number of mouse passaged xenograft lines from both B-ALL and T-ALL patients with varying cytogenetics. Thus, our data provides evidence that azacitidine and panobinostat can successfully overcome osteoblast-induced chemoprotection in vitro and ex vivo in both B-ALL and T-ALL cells.

Sensitization of acute lymphoblastic leukemia cells for LCL161-induced cell death by targeting redox homeostasis

Disturbed redox homeostasis with both elevated reactive oxygen species (ROS) levels and antioxidant defense mechanisms has been reported in acute lymphoblastic leukemia (ALL). We therefore hypothesized that inhibition of pathways responsible for ROS detoxification renders ALL cells more susceptible for cell death. Here, we report that pharmacological inhibitors of key pathways for the elimination of ROS, i.e. Erastin, buthionine sulfoximine (BSO) and Auranofin, sensitize ALL cells for cell death upon treatment with the Smac mimetic LCL161 that antagonizes Inhibitor of Apoptosis (IAP) proteins. Erastin, BSO or Auranofin significantly increase LCL161-induced cell death and also act in concert with LCL161 to profoundly suppress long-term clonogenic survival in several ALL cell lines. Erastin or BSO cooperates with LCL161 to stimulate ROS production and lipid peroxidation prior to cell death. ROS production and lipid peroxidation are required for this cotreatment-induced cell death, since ROS scavengers or pharmacological inhibition of lipid peroxidation provides significant protection against cell death. These results emphasize that inhibition of antioxidant defense mechanisms can serve as a potent approach to prime ALL cells for LCL161-induced cell death.

Targeting bone marrow lymphoid niches in acute lymphoblastic leukemia

In acute lymphoblastic leukemia (ALL) the bone marrow microenvironment provides growth and survival signals that may confer resistance to chemotherapy. Granulocyte colony-stimulating factor (G-CSF) potently inhibits lymphopoiesis by targeting stromal cells that comprise the lymphoid niche in the bone marrow. To determine whether lymphoid niche disruption by G-CSF sensitizes ALL cells to chemotherapy, we conducted a pilot study of G-CSF in combination with chemotherapy in patients with relapsed or refractory ALL. Thirteen patients were treated on study; three patients achieved a complete remission (CR/CRi) for an overall response rate of 23%. In the healthy volunteers, G-CSF treatment disrupted the lymphoid niche, as evidenced by reduced expression of CXCL12, interleukin-7, and osteocalcin. However, in most patients with relapsed/refractory ALL expression of these genes was markedly suppressed at baseline. Thus, although G-CSF treatment was associated with ALL cell mobilization into the blood, and increased apoptosis of bone marrow resident ALL cells, alterations in the bone marrow microenvironment were modest and highly variable. These data suggest that disruption of lymphoid niches by G-CSF to sensitize ALL cells to chemotherapy may be best accomplished in the consolidation where the bone marrow microenvironment is more likely to be normal.

Primary Acute Lymphoblastic Leukemia Cells Create a Drug-Resistant Niche By Tunneling Nanotube Signaling with Mesenchymal Stromal Cells

Background: Acute lymphoblastic leukemia (ALL) cells reside in the bone marrow microenvironment, which nurtures and protects cells from chemotherapeutic drugs. Bidirectional signaling between leukemic cells and their niche is shown to be essential for these processes. A major gap in our knowledge is the lack of insight into the functional mechanism regulating and controlling this crosstalk. Tunneling nanotubes (TNTs) have recently been reported as a novel mode of communication between eukaryotic cells, which can facilitate the transport of several types of cellular cargo.Objectives: This study aims to investigate the presence and functional importance of TNTs in the leukemic niche.Results: Here, we show for the first time that TNT signaling occurs between primary patient-derived leukemic cells and primary mesenchymal stromal cells using flow cytometry and time-lapse confocal microscopy. TNTs form within minutes after the start of co-culture and efficiently transfer lipophilic carbocyanine dye DiI from ALL cells towards MSCs. Dye transfer was significantly reduced when TNT signaling was inhibited using three independent experimental setups: actin inhibition, mechanical disruption through gentle shaking of cell cultures, and physical separation using a 3.0 µm pore-sized transwell system (4-fold, 5-fold and 35-fold reduction, respectively). In reciprocal experiments we also observed dye transfer via TNTs from MSCs to BCP-ALL cells, but the magnitude was strikingly less (175-fold, p ≤ 0.001). In addition, we show that leukemic cells use TNTs to modulate their microenvironment by directing non-malignant stromal cells to produce pro-survival cytokines. We observed patient-specific cytokine signatures after co-culture of primary ALL cells with different primary MSCs. For example, IP10/CXCL10 levels increased more than 1000-fold when patient ALL#1 cells were co-cultured with primary MSCs, but were undetectable in co-cultures from patient ALL#2 cells. Induction of all cytokines was dependent on TNT signaling, as TNT inhibition significantly lowered cytokine production (p ≤ 0.001). Importantly, we show that TNT signaling is functionally important for leukemic cell survival and stroma-driven drug resistance. Leukemic cell viability was assessed by flow cytometry after staining with Annexin V, Propidium Iodide and CD19. Primary leukemic cell survival significantly increased in 5-day co-cultures with primary MSCs compared to mono-culture (p ≤ 0.001). When TNT formation was prevented by shaking of these co-cultures or by transwell conditions, the cell viability reduced 3.5- and 3.6-fold, respectively (p ≤ 0.001). TNT inhibition similarly reduced the survival of primary ALL cells in co-culture with MSCs during prednisolone exposure (p ≤ 0.01). Co-culture with primary MSCs also induced resistance to prednisolone 2.5-fold compared to mono-culture in a proliferative setting, using the BCP-ALL cell line NALM6. Inhibition of TNT formation by shaking or transwell conditions significantly reduced this effect: cells in co-culture were only 1.4- and 1.1-fold more resistant compared to mono-culture, respectively (p ≤ 0.01).Conclusion: The presented study identifies TNT formation as a major regulator of interaction between ALL cells and their bone marrow niche, which facilitates signaling mainly from leukemic cells towards MSCs. This signaling drives the release of cytokines within the microenvironment. Disruption of tunneling nanotubes inhibits this release, diminishes the survival benefit that MSCs provide to primary ALL cells, and sensitizes ALL cells to the important anti-leukemic drug prednisolone. This observation gives insight into the pathogenesis of BCP-ALL and opens new avenues to develop more effective therapies that interfere with the leukemic niche. DisclosuresNo relevant conflicts of interest to declare.

Therapy-resistant acute lymphoblastic leukemia (ALL) cells inactivate FOXO3 to escape apoptosis induction by TRAIL and Noxa

Forkhead transcription factors (FOXO) are downstream targets of the phosphoinositol-3-kinase (PI3K) protein kinase B (PKB) signaling cascade and play a pivotal role in cell differentiation, cell cycle and apoptosis. We found that cells from prednisone-resistant T-acute lymphoblastic leukemia (T-ALL) patients showed cytoplasmic localization of FOXO3 in comparison to prednisone-sensitive patients suggesting its inactivation. To determine the impact of FOXO3, T-ALL cells were infected with a 4OH-tamoxifen-regulated, phosphorylation-independent FOXO3(A3)ERtm allele. After FOXO3-activation these cells undergo caspase-dependent apoptosis. FOXO3 induces the death ligand TRAIL and the BH3-only protein Noxa implicating extrinsic as well as intrinsic death signaling. Whereas dnFADD partially inhibited cell death, CrmA and dnBID efficiently rescued ALL cells after FOXO3 activation, suggesting a caspase-8 amplifying feedback loop downstream of FADD. Knockdown of TRAIL and Noxa reduced FOXO3-induced apoptosis, implicating that mitochondrial destabilization amplifies TRAIL-signaling. The-reconstitution of the cell cycle inhibitor p16INK4A, which sensitizes ALL cells to mitochondria-induced cell death, represses FOXO3 protein levels and reduces the dependency of these leukemia cells on PI3K-PKB signaling. This suggests that if p16INK4A is deleted during leukemia development, FOXO3 levels elevate and FOXO3 has to be inactivated by deregulation of the PI3K-PKB pathway to prevent FOXO3-induced cell death.

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

The CXCR4 antagonist Plerixafor (AMD3100) induces the rapid mobilization of hematopoietic stem and progenitor cells into the blood in mice and humans. AMD3100 similarly induces the mobilization of human acute lymphoblastic leukemia (ALL) cells into the blood in mice. In this study, the temporal response of pre-B ALL cells to AMD3100 was compared with that of normal hematopoietic progenitor cells (HPC) using an NOD/SCID xenograft model of ALL and BALB/c mice, respectively. ALL cells remained in the circulation up to 6 hours after AMD3100 administration, by which time normal HPCs were no longer detectable. AMD3100 also increased the proportion of actively cycling ALL cells in the peripheral blood. Together, these data suggest that ALL cells are more sensitive to the effects of bone marrow disruption than normal progenitors. Using the NOD/SCID xenograft model, we demonstrated that AMD3100 increased the efficacy of the cell cycle specific drug vincristine, resulting in reduced disease levels in the blood and spleens of animals over 3 weeks and extended the survival of NOD/SCID mice with ALL. These data demonstrate that mobilizing agents can increase the therapeutic effect of cell cycle dependent chemotherapeutic agents.

Targeting Aurora Kinases in Acute Lymphoblastic Leukemia

AbstractAbstract 4907 Introduction:Despite improved treatment outcome in acute lymphoblastic leukemia (ALL), drug resistance and disease recurrence remain major obstacles in specific subgroups. Thus, there is an urgent need to identify new targets for therapy. Several studies showed that Aurora kinases were therapeutic targets in cancer therapy, including solid tumors and hematological malignancies. Here we describe preclinical testing of Aurora kinase inhibitors in ALL and the molecular mechanism of different drug activity. Materials and methods:Quantitative RT-PCR and Western blot were used to assess the expressions of Aurora kinases and their activators in ALL. RT-PCR was used to detect the expression of MDR-1. To test activity against Aurora kinases, different ALL cell lines were treated with various concentrations of Aurora kinase inhibitors “VE-465 and VX-680”. The effects of Aurora kinase inhibitors on the cell cycle were evaluated by flow cytometry. Gene expression profiling was performed to identify the candidate targets which regulate the different drug sensitivity. Transient knockdown and overexpression of candidate genes in ALL cell lines were also employed in this study. Results:Nine ALL cell lines treated with Aurora kinase inhibitors (VE-465 and VX-680) exhibited different drug sensitivity. Five ALL cell lines were sensitive to drug treatment with IC50 around 10–40 nM, including MLL-AF4-positive and BCR-ABL-positive cell lines. However, RPMI-8402 was one of the three cell lines which were resistant to VE-465 and VX-680 with IC50 more than 10 μM. Among these sensitive ALL cell lines, treatment of Aurora kinase inhibitors resulted in an increased G2/M and sub-G1 populations. In contrast, drug-resistant ALL cell lines showed increased polyploidy status after exposure to Aurora kinase inhibitors. The different treatment efficacy was not related to the expression of Aurora kinases, their activators or MDR-1. In order to elucidate the molecular mechanism to regulate the different drug sensitivity, microarray study was performed. It showed that treatment of Aurora kinase inhibitors resulted in differential expressions of genes (75 up-regulated and 90 down-regulated genes) and CDKN1Awas one of the potential molecules which regulated the treatment diversity. RT-PCR and Western blot confirmed the cDNA microarray data: CDKN1A was up-regulated after treatment with Aurora kinase inhibitors in the drug-sensitive cell lines, but no change in the level of CDKN1A in the drug-resistance cell lines. Knockdown of CDKN1A in drug-sensitive cell lines impaired the treatment activity. Over-expression of CDKN1A in drug-resistant cell lines increased the anti-leukemia effect of Aurora kinase inhibitors. Conclusion:These data suggest that treatment with Aurora kinase inhibitors may be a novel and effective therapy in specific subgroups of ALL, including MLL-AF4-positive ALL. These data show that status of Aurora kinases, their activators or MDR-1 does not correlate with the drug susceptibility in ALL cell lines. The susceptibility to Aurora kinase inhibitors in ALL depends on the activation status of CDKN1A. Disclosures:No relevant conflicts of interest to declare.

Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic leukemia

Glucocorticoids (GCs) are among the most widely prescribed medications in clinical practice. The beneficial effects of GCs in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis, but the underlying transcriptional mechanisms remain poorly defined. Computational modeling has enormous potential in the understanding of biological processes such as apoptosis and the discovery of novel regulatory mechanisms. We here present an integrated analysis of gene expression kinetic profiles using microarrays from GC sensitive and resistant ALL cell lines and patients, including newly generated and previously published data sets available from the Gene Expression Omnibus. By applying time-series clustering analysis in the sensitive ALL CEM-C7-14 cells, we identified 358 differentially regulated genes that we classified into 15 kinetic profiles. We identified GC response element (GRE) sequences in 33 of the upregulated known or potential GC receptor (GR) targets. Comparative study of sensitive and resistant ALL showed distinct gene expression patterns and indicated unexpected similarities between sensitivity-restored and resistant ALL. We found that activator protein 1 (AP-1), Ets related gene (Erg) and GR pathways were differentially regulated in sensitive and resistant ALL. Erg protein levels were substantially higher in CEM-C1-15-resistant cells, c-Jun was significantly induced in sensitive cells, whereas c-Fos was expressed at low levels in both. c-Jun was recruited on the AP-1 site on the Bim promoter, whereas a transient Erg occupancy on the GR promoter was detected. Inhibition of Erg and activation of GR lead to increased apoptosis in both sensitive and resistant ALL. These novel findings significantly advance our understanding of GC sensitivity and can be used to improve therapy of leukemia.

Inhibition of Akt Potentiates 2-DG–Induced Apoptosis via Downregulation of UPR in Acute Lymphoblastic Leukemia

The ability to pair the regulation of metabolism and cellular energetics with oncogenes and tumor suppressor genes provides cancer cells with a growth and survival advantage over normal cells. We investigated the mechanism of cell death induced by 2-deoxy-D-glucose (2-DG), a sugar analog with dual activity of inhibiting glycolysis and N-linked glycosylation, in acute lymphoblastic leukemia (ALL). We found that, unlike most other cancer phenotypes in which 2-DG only inhibits cell proliferation under normoxic conditions, ALL lymphoblasts undergo apoptosis. Bp-ALL cell lines and primary cells exhibited sensitivity to 2-DG, whereas T-ALL cells were relatively resistant, revealing phenotypic differences within ALL subtypes. Cotreatment with D-mannose, a sugar essential for N-linked glycosylation, rescues 2-DG-treated ALL cells, indicating that inhibition of N-linked glycosylation and induction of ER stress and the unfolded protein response (UPR) is the predominant mechanism of 2-DG's cytotoxicity in ALL. 2-DG-treated ALL cells exhibit upregulation of P-AMPK, P-Akt, and induction of ER stress/UPR markers (IRE1α, GRP78, P-eIF2α, and CHOP), which correlate with PARP cleavage and apoptosis. In addition, we find that pharmacologic and genetic Akt inhibition upregulates P-AMPK, downregulates UPR, and sensitizes ALL cells to remarkably low doses of 2-DG (0.5 mmol/L), inducing 85% cell death and overcoming the relative resistance of T-ALL. In contrast, AMPK knockdown rescues ALL cells by upregulating the prosurvival UPR signaling. Therefore, 2-DG induces ALL cell death under normoxia by inducing ER stress, and AKT and AMPK, traditionally thought to operate predominantly on the glycolytic pathway, differentially regulate UPR activity to determine cell death or survival.

UNC569 As Novel Small Molecule Mer Receptor Tyrosine Kinase Inhibitor for Treatment of ALL

Abstract 2589 Introduction:Acute lymphoblastic leukemia (ALL) is one of the most common malignancies in children. Pediatric cure rates for ALL have dramatically increased in recent years as intensified therapeutic regimens have been developed. However, intensified therapy is associated with a significant and increased risk of short- and long-term toxicities. In addition, patients who relapse, do not reach remission, or have certain cytogenetic abnormalities have a poor prognosis and limited treatment options. Therefore, novel and less toxic therapies are needed. Tyrosine kinases are frequently abnormally regulated in cancer cells. Mer receptor tyrosine kinase is ectopically expressed in ALL cell lines and patient samples. Inhibition of Mer expression reduces pro-survival signaling, dramatically increases the sensitivity of leukemia cells to cytotoxic agents, and significantly delays development of leukemia in a mouse model. Thus, Mer tyrosine kinase inhibitors (TKIs) are excellent candidates for targeted therapies. We report here the first small molecule selective for Mer TK (UNC569) and characterization of its biochemical and anti-tumor activities in cell culture models of ALL. Methods:UNC569 is a substituted pyrazolopyrimidine that has been developed by a structure-based design and iterative medicinal chemistry from the known Mer/C52 cocrystal structure. Inhibition of Mer kinase activity by UNC569 was determined by a microfluidic capillary electrophoresis (MCE) assay in which phosphorylated and unphosphorylated substrate peptides were separated and analyzed through a LabChip EZ Reader. Western blot analysis of phosphorylated and total Mer protein was used to determine Mer inhibition by UNC569 in 697 (B-ALL) and Jurkat (T-ALL) cell lines. UNC569-mediated anti-leukemia activity was determined by detection of metabolically active cells using MTT reagent after 48 hours of exposure and by determining colony-formation in methylcellulose medium in the presence of UNC569. To investigate interactions with standard ALL therapies, apoptotic and dead cells were identified by flow cytometric analysis of cells stained with YO-PRO-1 and propidium iodide dyes after treatment with a chemotherapeutic agent alone or in combination with UNC569. Results:UNC569 is a novel small molecule Mer TKI with potent activity against Mer kinase (IC50 = 2.9 nM). In cell-based assays, UNC569 inhibited accumulation of phospho-Mer in both 697 and Jurkat ALL cells (IC50 <100 nM). Reduced proliferation and/or survival of ALL cells was also observed in 697 (IC50 = 0.91 ± 0.16 μM) and Jurkat (IC50 = 1.55 ± 0.19 μM) cultures. Treatment with UNC569 resulted in a statistically significant, dose-dependent decrease in colony-formation in Jurkat and 697 cell lines (60 ± 13.8 % (p = 0.01) and 29 ± 12.1 % (p = 0.01), respectively, relative to untreated cultures). Treatment of 697 cells with UNC569 in combination with etoposide resulted in a statistically significant increase in apoptotic and dead cells when compared with etoposide alone (43.3 ± 4.6 % vs 33.03 ± 0.73 %, p = 0.02). Treatment of Jurkat cells with a combination of UNC569 and methotrexate also resulted in a statistically significant increase in apoptotic and dead cells relative to methotrexate alone (26.9 ± 9.5 % vs 18.5 ± 5.7 %, p = 0.03). Conclusion:UNC569 is a highly effective Mer TKI that inhibits accumulation of the active phosphorylated form of Mer in B- and T-ALL cells. UNC569 mediates anti-leukemia activity against B- and T-ALL cells in culture and decreases colony-forming potential in methylcellulose. In addition, treatment with UNC569 sensitizes ALL cells to cytotoxic agents that are currently used as standard ALL therapies. Taken together, these data suggest that treatment with UNC569 may be a novel and effective ALL therapy and may be particularly effective in combination with cytotoxic therapies, thereby allowing for dose reduction and decreased incidence of toxic side effects. Disclosures:No relevant conflicts of interest to declare.

FOXO3a Mediates Both the Pro-Survival and Proapoptotic Effects of Glucocorticoids In Acute Lymphoblastic Leuekmia

AbstractAbstract 1822Synthetic glucocorticoids (GCs) such as dexamethasone and prednisone are the mainstay of acute lymphoblastic leukemia (ALL) therapy. However, in spite of extensive use of these compounds in treating lymphoid malignancies, the molecular events that lead to cell death upon GC exposure are only poorly understood. Elucidation of these events is crucial to decipher the mechanisms of resistance to GC seen commonly at the time of recurrent disease as well as in older patients diagnosed with ALL and to design therapy targeted to overcome GC resistance. We investigated the molecular events leading to cell death in a panel of ALL cell lines with varying sensitivity to dexamethasone induced cell death. Dexamethasone induced cell death in sensitive cell lines was caspase dependent, preceded by mitochondrial membrane depolarization and cytochrome C release. Since the mitochondrial integrity is controlled by the members of the BCL2 family of proteins, we evaluated the expression of various pro- and anti-apoptotic BCL2 family proteins in ALL cell lines treated with dexamethasone and found a consistent upregulation of Bim, a BH3-only proapoptotic BCL2 family protein. This was accompanied by accumulation of non-phosphorylated FOXO3a, a member of the forkhead transcription factors known to be involved in transcriptional regulation of Bim expression. This accumulation of non-phosphorylated FOXO3a was associated with increased reporter activity in a FOXO-responsive luciferase construct, increased luciferase reporter activity in the Bim promoter containing the canonical foxo response element, and increased occupancy of FOXO3a in the Bim promoter as demonstrated by chromatin immunoprecipitation. However, shRNA mediated partial knock-down of FOXO3a did not rescue ALL cells from dexamethasone induced cell death. Indeed, this partial reduction in FOXO3a levels resulted in sensitization of ALL cells to dexamethasone induced cell death. Similar to the parental or control transfected cells, dexamethasone induced cell death in ALL cells with reduced FOXO3a expression could be inhibited by the pan-caspase inhibitor z-VAD-fmk. Further, the partial reduction in FOXO3a expression did not interfere with Bim up-regulation in response to GC exposure. In addition to modulating apoptosis by transcriptional regulation of molecules like Bim, FOXO family members are also involved in regulating cellular reactive oxygen species (ROS), by transcriptional regulation of molecules like manganese-superxode dismutase involved in ROS scavenging pathways. We therefore measured ROS by dihydroethidium staining in control and FOXO3 shRNA transfected ALL cell lines in the presence or absence of dexamethasone exposure. We found no difference in the ROS levels in the non-treated cells. However, upon exposure to dexamethasone, cells with lower FOXO3a levels demonstrated higher accumulation of ROS when compared to the control cell lines. We conclude that accumulation of non-phosphorylated, active FOXO3a in response to GC exposure mediates both the pro-survival and proapoptotic response in ALL cells and that selective abrogation of the antioxidant, pro-survival functions of FOXO3a might provide a feasible strategy to sensitize ALL cells to GC mediated cell death. Disclosures:No relevant conflicts of interest to declare.

Abstract 2911: Small molecule XIAP inhibitors sensitize acute leukemia cells for TRAIL- or chemotherapy-induced apoptosis, overcome Bcl-2-mediated resistance and reduce leukemic burden in vivo in NOD/SCID mice

Abstract Defects in apoptosis contribute to poor outcome in high risk pediatric acute lymphoblastic leukemia (ALL), calling for novel strategies that counter apoptosis resistance. XIAP, a member of “Inhibitor of Apoptosis” (IAP) proteins, presents a promising target, since it is expressed at high levels in acute leukemia. Therefore, we investigated the effect of small molecule XIAP inhibitors alone and in combination with TRAIL or chemotherapeutics in ALL cell lines, primary leukemic blasts, normal peripheral blood lymphocytes and in a mouse model of pediatric ALL. Here, we report that XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, cooperate with TRAIL to induce apoptosis in ALL cells in a highly synergistic manner as calculated by combination index. Also, XIAP inhibitors act in concert with TRAIL to reduce clonogenic growth of ALL cells demonstrating that they suppress longterm survival. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner, indicating that they promote a caspase-dependent mitochondrial amplification loop. Intriguingly, XIAP inhibitors overcome Bcl-2-mediated resistance to TRAIL by promoting Bcl-2 cleavage, Bak conformational change and caspase-3-driven mitochondrial perturbations. Thus, the combination of XIAP inhibitors and TRAIL can even break Bcl-2-imposed resistance, a defect in the apoptotic pathway that frequently occurs in acute leukemia and predicts poor prognosis. Moreover, XIAP inhibitors sensitize ALL cells for chemotherapy, e.g. cytarabine, doxorubicin, etoposide and 6-mercaptopurine. Intriguingly, this chemosensitization is inhibited by a TNFalpha-neutralizing antibody, demonstrating that it depends on XIAP inhibitor-triggered production of TNFalpha. Notably, XIAP inhibitors potently trigger cell death in leukemic blasts from children with ALL ex vivo and also sensitize primary leukemia cells for TRAIL- or chemotherapy-induced cell death. In contrast to malignant cells, XIAP inhibitors and TRAIL at equimolar concentrations are non-toxic to normal peripheral blood lymphocytes despite expression of the apoptosis-inducing TRAIL receptors on the cell surface, pointing to a therapeutic window. Most importantly, XIAP inhibitors exert potent anti-leukemic activity in vivo in a mouse model of pediatric ALL engrafted in NOD/SCID mice. These findings provide first evidence that XIAP inhibitors present a novel strategy to trigger apoptosis in childhood leukemia and to prime acute leukemia cells for TRAIL- or chemotherapy-induced cell death. These data have important clinical implications for the development of apoptosis targeted therapies for childhood leukemia. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2911.

Small molecule XIAP inhibitors sensitize childhood acute leukemia cells for CD95‐induced apoptosis

Escape of apoptosis may contribute to treatment failure in childhood acute lymphoblastic leukemia (ALL) calling for new approaches to overcome apoptosis resistance. Here, we provide for the first time evidence that small molecule inhibitors that target the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) sensitize ALL cells for CD95-induced apoptosis. XIAP inhibitors at subtoxic concentrations, but not a structurally related control compound, act synergistically with agonistic anti-CD95 antibodies or MegaFasL, a hexameric form of CD95 ligand, to induce apoptosis in ALL cells. Further, XIAP inhibitors co-operate with MegaFasL to reduce clonogenic survival of ALL cells demonstrating their effect also on long-term survival. In contrast, XIAP inhibitors show little effect on MegaFasL-mediated apoptosis in normal peripheral blood lymphocytes (PBLs), pointing to some tumor selectivity. Molecular studies reveal that XIAP inhibitors enhance CD95-induced activation of caspases, loss of mitochondrial membrane potential and cytochrome c release in a caspase-dependent manner. Importantly, XIAP inhibitors sensitize primary leukemic blasts from children with ALL for MegaFasL-induced apoptosis. Thus, small molecule XIAP inhibitors present a promising novel approach to enhance CD95-induced apoptosis in childhood acute leukemia.

AKT inhibitor, GSK690693, induces growth inhibition and apoptosis in acute lymphoblastic leukemia cell lines

AbstractThe PI3K/AKT signaling is activated in various hematologic malignancies. We evaluated the effect of a novel, pan-AKT kinase inhibitor, GSK690693, on the proliferation of 112 cell lines representing different hematologic neoplasia. Fifty-five percent of all cell lines tested were sensitive to AKT inhibitor (EC50 < 1 μM), with acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, and Burkitt lymphoma showing 89%, 73%, and 67% sensitivity to GSK690693, respectively. The antiproliferative effect was selective for the malignant cells, as GSK690693 did not inhibit the proliferation of normal human CD4+ peripheral T lymphocytes as well as mouse thymocytes. Phosphorylation of downstream substrates of AKT was reduced in both sensitive and insensitive cell lines on treatment with GSK690693, suggesting that the cause of resistance was not related to the lack of AKT kinase inhibition. Consistent with the role of AKT in cell survival, GSK690693 also induced apoptosis in sensitive ALL cell lines. Overall, our data provide direct evidence for the role of AKT signaling in various hematologic malignancies, especially ALL and some lymphomas.

Up-regulation of c-FLIPS+R upon CD40 stimulation is associated with inhibition of CD95-induced apoptosis in primary precursor B-ALL

Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.