SUP-B15 Cells Research Articles

The Novel Phospholipid Gemcitabine Conjugate KPC34 Has Activity In Acute Leukemia

Abstract Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are cancers of the bone marrow that in adults, occur mainly in the elderly. About 19,000 Americans develop these diseases annually and the 5 year survival rate is 30-40%. The prognosis for elderly patients is even worse with a 5 year survival rate of 10%. Treatment for AML and ALL involves induction chemotherapy using the deoxycytidine analog cytarabine followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Recurrent disease is much more difficult to treat successfully and elderly patients often have trouble tolerating repeated chemotherapy treatments. Chemoresistance can occur by several mechanisms including reduced activation of cytarabine via downregulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. Low expression of both dCK and ENT-1 correlate with poor prognosis in patients with acute leukemias. Another contributor to cytarabine resistance is the interaction of leukemia cells with bone marrow stroma. This interaction confers protection to leukemia cells from cytarabine by inhibiting apoptosis and ENT-1 function. Additionally, activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms α, β1, and β2 have been detected in both AML and ALL and promote leukemic proliferation and survival by activation of ERK and the anti-apoptotic protein BCL-2. The development of novel therapeutic agents or modifying existing drugs may be able to overcome chemoresistance in AML and ALL and improve clinical outcomes. KPC34 is a novel phospholipid conjugate of the deoxycytidine analog gemcitabine. Gemcitabine acts similarly to cytarabine and is administered IV to treat various tumors and lymphomas, but is rarely used for leukemias. Compared to cytarabine, gemcitabine treatment inhibits DNA replication more effectively, prevents repair of damaged DNA, and uniquely inhibits ribonucleotide reductase. KPC34's novel properties result in improved pharmacokinetics compared to gemcitabine and may be able to overcome leukemic chemoresistance to cytarabine by bypassing the need for ENT-1 uptake and dCK activation. In addition, KPC34 can be administered orally, is predicted to cross the blood-brain barrier, allowing it to target CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety is predicted to inhibit the activity of PKC. In order to determine if KPC34 has activity against acute leukemias we tested it against a variety of human and murine leukemic cell lines in vitro. KPC34 was highly cytotoxic to with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). Exposure of leukemia cells to KPC34 resulted in the induction of apoptosis as assessed by annexin V and PI assays. Human leukemic cell lines treated with KPC34 had dose-dependent reduction of colony formation, where normal donor hematopoietic stem cells did not. To test the ability of KPC34 to overcome stroma-mediated chemoresistance, SUPB15 cells were incubated in the presence or absence of a bone marrow stroma cell line for 48 hrs. The cells were then treated with 10 nM cytarabine or KPC-34. No significant difference was seen between either treatment without stroma, while KPC34 significantly increased apoptosis in the presence of stroma (p value ≤ 0.001, by Student’s t test). In order to assess the activity of KPC34 in vivo, we injected C57/BL6 mice with a syngeneic, murine Ph+ pre-B cell ALL, luciferase expressing cell line. When engraftment was established by bioluminescence imaging, mice were treated with 25 mg/kg KPC34 or water by oral gavage on days 1, 4 and 7. Treatment resulted in a significant prolongation of survival with treated mice having a median survival of 26 days compared to 13 days for controls (p value of 0.0042, by log-rank test). These data demonstrate the activity of KPC34 in multiple preclinical models of leukemia and support its continued translation to the clinic. Disclosures: Kucera: Wake Forest Baptist Health: KPC 34 Patent, KPC 34 Patent Patents & Royalties.

Evaluation Of IKZF1 Δ4-7 Deletion As a Suitable Marker For Minimal Residual Disease Monitoring; A Study Of 161 Consecutive Acute Lymphoblastic Leukaemia (ALL) Patients On The On-Going UKALL14 Trial

Minimal residual disease (MRD) testing is vital to risk assignment in acute lymphoblastic leukaemia (ALL). Quantification of patient-specific rearrangements of immunoglobulin and T-cell receptor (Ig/TCR) genes is the most standardised method in current use. Recent studies demonstrated that deletion in the IKZF1 gene is common and is prognostic of poor outcome. IKZF1 targets could offer potential for MRD monitoring in BCR-ABLnegative (neg) ALL. The 5' and 3' break points of this deletion are highly conserved, making it possible to design a universal PCR assay to amplify the fusion genomic sequence created by the deletion.With the initial aim of developing an MRD assay we studied the incidence of IKZF1 Δ 4-7 in 161 consecutive patients enrolled on the on-going adult ALL trial UKALL14, aged 25-59 years. We carried out PCR using breakpoint-specific primers designed as close as possible to the breakpoint. In order to ensure that the screening PCR enabled sensitive detection of the deletions, serial dilution of a positive control SUP-B15 cell line diluted into non-tumoral DNA was analysed. IKZF1 Δ 4-7 could be detected with a sensitivity of at least 10-4 (0.01%). PCR bands could be readily allocated to “strong” or “weak” on visual inspection; all PCR positive signals were confirmed as IKZF1 Δ 4-7 by Sanger sequencing of the PCR products and the breakpoints mapped. The frequency of the deletion in the total population studied was 80/161 (50%); 23/35 (66%) in the BCR-ABL positive (pos) subgroup and strikingly 57/126 (45%) in the BCR-ABLneg subgroup. The high rate of IKZF1 Δ 4-7 in the BCR-ABLneg patients was unexpected, being approximately double the reported incidence BCR-ABLneg adults.Real time quantitative (RQ)-PCR analyses to investigate the suitability of IKZF1 quantitation as the basis for an MRD assay was performed on 26 BCR-ABLneg cases, selected only on the basis of availability of follow-up material. Assays were assessed using the same criteria applied by EuroMRD for Ig/TCR quantification. On that basis, “limited testing” of PCR amplifications at the 10-2 (1%) and 10-4(0.01%) dilution points was performed on all 26 samples. To our surprise, only 5 samples gave acceptable data to qualify for a quantitative MRD assay. Notably, all the 21 cases that failed “limited testing” had yielded a weak PCR signal on initial screening, suggesting the PCR assay have detected intragenic deletions restricted to minor subclones.Although subclonal IKZF1 gene alterations are well described, the apparent high frequency of these events in our cohort (21/26 tested for RQ-PCR assay) was surprising. To further analyse this, we performed MRD-based genomic quantification of all 26 diagnostic specimens using SUP-B15 dilution series. The percentage of ALL cells bearing IKZF1 Δ 4-7 in the putative 21 subclonal cases was <0.01% compared to >90% in the putative 5 major clonal cases. In total, 82% of BCR-ABLneg cases gave a weak PCR band at initial screening, suggesting that most of the Δ 4-7 in BCR-ABLneg adult ALL are subclonal.In the 5 cases where a quantifiable IKZF1 Δ 4-7-based MRD assay could be developed, MRD analysis was performed on 12 follow-up samples and sensitivity and quantitative range of at least 10-4 (0.01%) and 5×10-4 (0.05%) were obtained, respectively. These parameters compare favourably with the performance of standard Ig/TCR assays. Notably in one sample MRD could only be quantified by the IKZF1 deletion approach (level= 7.79E-05, ∼ 0.008%).IKZF1 lesions are not leukaemia-initiating events, hence the stability of this alteration during the history of ALL is not clear. To address this, 14 diagnosis and relapse paired samples were analysed. Three different patterns emerged: in 5 cases the IKZF1 Δ 4-7 at diagnosis was preserved at relapse; in 3 cases the lesion was newly acquired at relapse; and in 3 cases the deletion was lost at relapse, notably all these 3 cases had showed a weak PCR signal, suggesting the deletion which was lost at relapse was subclonal at diagnosis. Theoretically this observation suggests that IKZF1deletion-based MRD monitoring carries a risk of missing a resurgent clone.In conclusion, IKZF1 Δ 4-7 can provide highly sensitive MRD assays and could be a potential candidate to add to the repertoire of currently available MRD markers. However, we have shown limited applicability due to many IKZF1 deletions occurring in putative subclones. The stability of these deletions in major clones remains to be determined. Disclosures:No relevant conflicts of interest to declare.

Retargeting NK-92 cells by means of CD19- and CD20-specific chimeric antigen receptors compares favorably with antibody-dependent cellular cytotoxicity

Multiple natural killer (NK) cell-based anticancer therapies are currently under development. Here, we compare the efficiency of genetically modified NK-92 cells expressing chimeric antigen receptors (CARs) at killing NK cell-resistant B-lymphoid leukemia cells to the antibody-dependent cell-mediated cytotoxicity (ADCC) of NK-92 cells expressing a high affinity variant of the IgG Fc receptor (FcγRIII). First, we compared in vitro the abilities of NK-92 cells expressing CD20-targeting CARs to kill primary chronic lymphocytic leukemia (CLL) cells derived from 9 patients with active, untreated disease to the cytotoxicity of NK-92 cells expressing FcγRIII combined with either of the anti-CD20 monoclonal antibodies (mAbs) rituximab or ofatumumab. We found that CAR-expressing NK-92 cells effectively kill NK cell-resistant primary CLL cells and that such a cytotoxic response is significantly stronger than that resulting from ADCC. For studying CAR-expressing NK cell-based immunotherapy in vivo, we established xenograft mouse models of residual leukemia using the human BCR-ABL1+ cell lines SUP-B15 (CD19+CD20−) and TMD-5 (CD19+CD20+), two acute lymphoblastic leukemia (ALL) lines that are resistant to parental NK-92 cells. Intravenous injection of NK-92 cells expressing CD19-targeting CARs eliminated SUP-B15 cells, whereas they had no such effect on TMD-5 cells. However, the intrafemoral injection of NK-92 cells expressing CD19-targeting CAR resulted in the depletion of TMD-5 cells from the bone marrow environment. Comparative studies in which NK-92 cells expressing either CD19- or CD20-targeting CARs were directly injected into subcutaneous CD19+CD20+ Daudi lymphoma xenografts revealed that CD20-targeting CAR is superior to its CD19-specific counterpart in controlling local tumor growth. In summary, we show here that CAR-expressing NK-92 cells can be functionally superior to ADCC (as mediated by anti-CD20 mAbs) in the elimination of primary CLL cells. Moreover, we provide data demonstrating that the systemic administration of CAR-expressing NK-92 cells can control lymphoblastic leukemia in immunocompromised mice. Our results also suggest that the direct injection of CAR-expressing NK-92 cells to neoplastic lesions could be an effective treatment modality against lymphoma.

In Vitro and in Vivo Single-Agent Efficacy of Checkpoint Kinase 1 (Chk1) and 2 (Chk2) Inhibitor PF-0477736 (Pfizer) in B- and T-Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 1496 Introduction:Although progress in the treatment of ALL has been remarkable in children, in adults ALL still carries a dismal outcome. Thus, there is a need to improve therapeutic options. In the last years, selective inhibitors of Chk1 and/or Chk2 have been discovered, developed and entered in clinical trials. However, so far, they have not yet been investigated in leukemia. Chk1 and Chk2 are serine/threonine kinases that play a critical role in response to DNA damage both by halting the cell cycle through checkpoint activation and by actively repairing DNA. Here, we explored the in vitro and in vivo activity of single-agent inhibition of Chk1/2 by PF-0477736 in B- and T-progenitor ALL and we investigated potential biomarkers of functional inhibition. Methods:Human B (BCR-ABL1-positive: BV-173, SUPB-15; BCR-ABL1- negative: NALM-6, NALM-19, REH) and T (MOLT-4, RPMI-8402, CEM) leukemia cell lines were incubated with increasing concentrations of drug (5–2000 nM) for 24, 48 and 72 hours (hrs). Results:Inhibition of Chk1/2 resulted in a dose and time-dependent cytotoxicity with RPMI-8402 and BV-173 cells being the most sensitive (IC50 at 24 hrs: 57 nM and 82 nM, respectively), while NALM-6 cells the most resistant (IC50 at 24 hrs: 1426 nM)(WST-1 assay, Roche). Sensitivity did not correlate with p53 status (BV-173, SUPB-15, NALM-6 and NALM-19 cells were p53 wild-type whereas REH, MOLT-4, RPMI-8402 and CEM cells were p53 mutated) and with baseline levels of Chk1/2 and ATR/ATM phosphorylation, indicative of intrinsic genetic stress. Consistent with the viability results, Annexin V/Propidium Iodide (PI) staining analysis showed a significant increase of apoptosis at 24 and 48 hrs in a dose and time dependent manner coupled to increased proteolytic cleavage of PARP-1. In all sensitive cell lines in addition to the induction of apoptosis, Chk1/Chk2 inhibition induced DNA damage as demonstrated by the increased number of γH2AX foci (western blot and immunofluorescence analysis) and by a marked phosphorylation of Chk1 (ser317 and ser345). Moreover, PF-0477736 efficiently triggered the Chk1-Cdc25-Cdk1 pathway as soon as 24 hrs of treatment with a decrease of the inhibitory phosphorylation of Cdc25c (ser216) and Cdk1 (tyr15), leading to the abrogation of cell cycle arrest as confirmed by PI staining analysis at 6 and 24 hrs. The efficacy of PF-0477736 was thereafter demonstrated in primary leukemic blasts separated from 14 ALL patients. Based on the viability results at 24 hrs, 3 groups of patients were identified: very good responders, 5/14, 36% (IC50: 100–500 nM); good responders, 6/14, 43% (IC50: 600–1000 nM); poor responders, 3/14, 21% (IC50 > 1000 nM). By contrast, PF-0477736 did not show efficacy in primary cultures of normal bone marrow mononuclear cells, demonstrating its specificity for leukemia cells. We extended the in vitro and ex-vivo studies by assessing the efficacy of Chk inhibition in mice transplanted with T-lymphoid leukemia, demonstrating that PF-0477736 increases the survival of treated mice compared with mice treated with vehicle (p = 0.0016). Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on treated leukemia cells and their untreated counterparts (DMSO 0.1%) after 24 hrs of incubation with concentrations equal to the IC50. Treatment resulted in a differential expression (p < 0.05) of genes involved in chromatin assembly, nucleosome organization and DNA packaging (e.g. Histone H1-H2A, 2B family clusters), DNA damage (DDIT3, GADD34 and GADD45a) and apoptosis (e.g. CDKN1A, BAX, FAS, BTG1), confirming that PF-0477736 contributes to checkpoint replication abrogation, accumulation of DNA damage and subsequent apoptosis in leukemia cells. Interestingly, N-Myc and c-Myc expression strongly decreased after treatment, as also confirmed by western blot analysis, suggesting that a negative feedback loop may exist between Chk induction and Myc expression. Conclusions:Together, these results demonstrate the efficacy of PF-0477736 both in vitro and in vivo models of ALL, arguing in favor of its future clinical evaluation in leukemia. Supported by ELN, AIL, AIRC, Fondazione Del Monte di Bologna-Ravenna, PRIN2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures:Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Oridonin in combination with imatinib exerts synergetic anti-leukemia effect in Ph+ acute lymphoblastic leukemia cells in vitro by inhibiting activation of LYN/mTOR signaling pathway

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by constitutively activated BCR-ABL and SRC family tyrosine kinases.They account for the activations of multiple growth-signaling pathways, including Raf/MEK/ERK, Akt/mTOR and STAT5 pathways. The BCR-ABL tyrosine kinase inhibitor imatinib is the standard treatment for Ph+ leukemia and plays efficacious role in CML. However, imatinib has few inhibitory effects on SRC tyrosine kinase with response rate of Ph+ ALL lower and relapse more frequent and quicker compared with CML. Previous studies showed that oridonin inhibits proliferation and induces apoptosis in many tumor cells. However, the anticancer activity and mechanism of oridonin in Ph+ ALL is unknown. To investigate the anticancer activity of oridonin, we examined its role in constitutively activated Akt/mTOR, Raf/MEK/ERK, STAT5 and SRC pathway, mRNA level of bcr/abl gene, cell viability and apoptosis in Ph+ ALL SUP-B15 cells. Furthermore, we detected synergetic effect of oridonin plus imatinib. Our results showed that oridonin inhibiting activations of LYN (one of SRC family kinases) and ABL and their downstream Akt/mTOR, Raf/MEK/ERK and STAT5 pathways, downregulated Bcl-2 but upregulated Bax protein and then induced apoptosis in Ph+ ALL cells. Oridonin plus imatinib exerted synergetic effects by overcoming imatinib defect of upregulating Akt/mTOR and LYN signaling. Additionally, we examined the effect of oridonin on the signaling pathways in the primary specimens from Ph+ ALL patients. Our data showed that oridonin remarkably suppressed activations of Akt/mTOR, Raf/MEK and STAT5 pathway in these primary specimens and oridonin with imatinib exerted synergetic suppressive effects on mTOR, STAT5 and LYN signaling in one imatinib resistant patient specimen. Additional evaluation of oridonin as a potential therapeutic agent for Ph+ ALL seems warranted.

Abstract B57: Overcoming Acquired Resistance in Philadelphia-Positive (Ph+) Leukemia

Abstract Chronic myeloid leukemia (CML) is characterized by the presence of p210Bcr-Abl which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors (AKIs) have been successfully established for the treatment of CML. Despite high rates of clinical response, CML patients can develop resistance. Acquired resistance to AKIs in Ph+ leukemia is mostly due to the acquisition of point mutations within the Bcr-Abl Kinase by either reducing the affinity of the kinase domain to the ATP-competitor or stabilizing the active confirmation of the Abl kinase domain. Thus, acquired drug resistance in Philadelphia positive (Ph+) leukemia and in particular the gatekeeper mutation, T315I, to Abl kinase inhibitors (AKIs), represents a major obstacle in overcoming Bcr-Abl positive leukemia. T315I mutation is refractory to clinically available AKIs, as well as to Abl allosteric inhibitors such as GNF-2. In this study, we documented cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between dasatinib and GNF-2. Cooperation was also documented in the ALL SupB15 cell line. Cooperation in cells carrying native Bcr-Abl gene was mediated by Bcr-Abl. In contrast, proliferation and clonigenicity inhibition of Ph+ cells harboring the T315I mutation was not mediated by Bcr-Abl, but apparently by Bcr-Abl-independent mechanisms. Moreover, previously we have identified oleic acid as the active component in the mushroom Daedalea gibbosa who inhibited the kinase activity of Bcr-Abl apparently by allosteric inhibition. Here, we report that derivatives of oleic acid, oleylamine-carbonyl L-valinol and oleylamine-carbonyl D-valinol, inhibited the activity of the native and T315I mutated Bcr-Abl. Furthermore, oleylamine-carbonyl L-valinol and oleylamine-carbonyl D-valinol, exhibited higher activity toward the oncogenic Bcr-Abl in comparison to native c-Abl in SupB15, Ph-positive ALL cell line.

The study of resistant mechanisms and reversal in an imatinib resistant Ph+ acute lymphoblastic leukemia cell line

In this study, we established an imatinib resistant Ph+ acute lymphoblastic leukemia (ALL) cell line SUP-B15/RI in vitro and studied the mechanism of imatinib resistance. Our results showed that the BCR-ABL1 fusion gene and the mdr1 gene were 6.1 times and 1.7 times, respectively, as high as that of parental SUP-B15 cell line. We found no mutation in the Abl kinase domain of SUP-B15/RI. Furthermore, the detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κB, JNK and STAT showed the up-regulation of phosphorylation of AKT, mTOR, P70S6K, and RAF, ERK, and MEK, down-regulation of PTEN and 4EBP-1, and no change in other cell signaling pathways in SUP-B15/RI. However, dasatinib and nilotinib showed partial resistance. Interestingly, bortezomib had no resistance. Imatinib combination with rapamycin had synergistic effect on overcoming the resistance. Altogether, over-expression of BCR-ABL1 and mdr1 gene were involved in the resistance mechanisms, and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line may be correlated with them. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinase, dasatinib, and nilotinib, not bortezomib. The combination of imatinib with rapamycin can partially overcome the resistance and blockade of the ubiquitin–proteasome can be also a promising pathway to overcome imatinib resistance.

5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype

Although the overall prognosis in childhood acute lymphoblastic leukaemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterised by the occurrence of disease at extramedullary sites such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofoetal antigen transcript levels in cytogenetic high-risk subgroups of patients (p < 0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4 positive leukemic blasts than healthy donors (p = 0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4+ (Sup5T4) and 5T4− (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared to Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4 positive Sup-B15 cells are susceptible to 5T4 specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high risk pre-B-ALL.

The Novel Small Molecule Chk1/Chk2 Inhibitor PF-0477736 (Pfizer) Is Highly Active As Single Agent in Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ ALL)

Abstract 76Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases regulated by Ataxia-Telangiectasia and Rad3-related (ATR) kinases and involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Deregulation of these pathways has been previously described in BCR-ABL positive cells and involved in chemoresistance. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, in this study we aimed to investigate the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in Ph+ ALL and to determine potential biomarkers of functional inhibition.We first examined Chk1 and Chk2 mRNA expression levels in 45 newly diagnosed Ph+ ALL patients, in their paired remission samples, in 14 relapsed cases and in 3 Ph+ cell lines (BV-173, SUPB-15 and K562) by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation). Higher transcript levels of Chk1 but not Chk2 were found in newly diagnosed patients compared to remission samples (p = 0.0009 for Chk1 and p= 0.8183 for Chk2). Chk1 transcript levels were comparable between diagnosis and relapsed cases (p = 0.5728), suggesting that the ATR-Chk1 pathway is strongly activated in Bcr-Abl-positive cells. This was confirmed by phosphorylation of Chk1 Ser317 by western blotting analysis in Ph+ cell lines.We then evaluated the effect of PF-0477736 as single agent on cell viability using the Cell Proliferation Reagent WST-1 (Roche). BV-173, SUPB-15 and K562 cell lines were incubated with increasing concentration of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1–0.5 μM, with BV-173 being the most sensitive, while K562 the most resistant. These results were confirmed in primary blasts cells from a Ph+ ALL patient with wild-type Bcr-Abl and from 3 cases harboring the T315I Bcr-Abl mutation found to be insensitive to the available TKIs (IC50 ranged from 0.1–0.5 μM at 48 hours). Consistent with the WST-1 results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in both cell lines and primary cells. To test whether increased apoptosis resulted from Chk1 inhibition, we assessed the changes in phosphorylation of Cdc25c phosphatase, which is inactivated by Chk1 to prevent mitotic entry, and of γH2AX, which is increased in response to DNA damage. Western blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 and increases levels of γH2AX. Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening by amplification and subsequent sequencing of all coding exons of p53. All cell lines except for K562 and primary leukemia cells lacked mutations in the p53 gene, demonstrating that in Ph+ ALL PF-0477736 is highly effective also on p53 wild-type tumor cells.Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on 3 treated Ph+ cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of 211 genes including those involved in apoptosis and cell cycle (CEBPB, CUL1, Histone H1-H2A, 2B family clusters, Histone H4, DHX15, SNCB, FOS) and DNA damage, such as DNA-damage-inducible transcript 3 (DDIT3) and growth-arrest and DNA damage-inducible proteins GADD34 and GADD45a, suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in Ph+ ALL cells.In conclusion, for the first time we demonstrate in a large cohort of Ph+ ALL patients an over-expression of Chk1, providing a strong rational for its inhibition. In vitro treatment of Ph+ ALL cells with PF-0477736 alone resulted in reduction of inhibitory phosphorylation of Cdc25c, inhibition of proliferation and induction of biomarkers of DNA damage and apoptosis, suggesting that single-agent Chk1/2 inhibition may be an effective treatment strategy for Ph+ ALL.Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures:Baccarani:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract C71: Overcoming mechanism(s) of resistance to active-site TOR inhibitors in leukemia and lymphoma.

Abstract Introduction: The PI3K/AKT/TOR signaling network has been the focus of intense research in the cancer field as its activity is commonly elevated across cancers including leukemia and lymphoma. As an integral effector of the network, the target of rapamycin (TOR) functions as the catalytic subunit of two distinct protein complexes, TORC1 and TORC2. Both complexes promote survival of hematological malignancies through protein translation (TORC1) and AKT activation (TORC2). Unlike the classical allosteric TOR inhibitors (e.g. rapamycin), a new class of active-site TOR inhibitors (asTORi) more completely inhibits TOR enzymatic function in both TORC −1 and −2 complexes. TORC2 inhibition correlates with accumulation of FOXO transcription factors in the nucleus. Our lab showed that asTORi kill mouse leukemia and inhibit proliferation of human leukemia cells in vitro and in vivo. Here we investigated mechanisms of resistance to asTORi-mediated cell death in a human leukemia as well as a panel of human lymphoma cell lines. We show that asTORi induce nuclear localization of FOXO and transcription of cytostatic (i.e. p27) but not pro-apoptotic (ex. TRAIL) FOXO target genes. Combining asTORi with pharmacologically achievable concentrations of the histone deacetylase inhibitor (HDACi) upregulates transcription of TRAIL and dramatically enhances death of human leukemia cells in vitro. Similarly, asTORi potentiate a small molecule antagonist of the anti-apoptotic Bcl-2 family proteins whose expression is commonly elevated in lymphoma. Material and Methods: Cell Lines and reagents: The mouse p190 and human SUP-B15 cell lines are Ph+ B precursor acute lymphoblastic leukemia cells. The human lymphoma cell lines OCI-LY1, SUDHL-4, SUDHL-6 and RCK8 are germinal-center diffuse large-B-cell non-Hodgkins lymphoma (DLBCL). The HDACi (Vorinostat) and BCL-2 antagonist (ABT-263) were purchased from Merck &amp; Co and Selleck. The active-site TOR inhibitors were generously provided by Intellikine Inc. Cell cycle and death analysis: Flow cytometry was used in conjunction with 7-amino actinomycin D or annexin V staining along with forward scatter profile to assay cell viability. Flow cytometry and propidium iodide staining was used to measure DNA content for cell cycle analysis. qPCR: RNA was isolated using trizol method and analyzed by real time PCR. Subcellular localization: SUP-B15 and OCI-LY1 cell lines were fractionated into cytosolic and nuclear fraction then probed with anti-FOXO1 antibody. Anti -actin and -histone antibodies were used to confirm the purity of the fractions. Conclusion:The human leukemia cell line SUP-B15 and DLBCL cell lines survive inhibition of TOR by asTORi.asTORi promotes nuclear localization of FOXO and transcription of the cytostatic effector and FOXO target gene, p27.Combining HDACi with asTORi promotes transcription of TRAIL, a pro-apoptotic FOXO target gene, and markedly enhances apoptosis in SUP-B15 cells.asTORi potentiates cytotoxic effects of the BCL-2 inhibitor in DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C71.

Effects of Metal Salophene and Saldach Complexes on Lymphoma and Leukemia Cells

Schiff base transition metal complexes are an important class of compounds with great potential for therapeutic interventions. However, data on antileukemic and antilymphoma effects of these complexes are limited. The activity of N,N'-bis(salicylidene)-1,2-phenylenediamine (salophene, 1), its iron(II/III) and manganese(II/III) complexes as well as rac-trans-N,N'-bis(salicylidene)-1,2-cyclohexanediamine (saldach, 2) and its respective iron(II/III) complexes was evaluated against U-937 non-Hodgkin's lymphoma and the HL-60, SUP-B15, and K-562 leukemia cell lines. The free ligands induced in all cell lines, if at all, only marginal, concentration-dependent growth inhibitory effects, and did not trigger Cu/Zn superoxide dismutase (Cu/Zn SOD) release or induce apoptosis. [Fe(II) (salophene)] (3) and [Fe(III) (salophene)Cl] (4) blocked cellular growth, caused a strong release of Cu/Zn SOD and induced apoptosis. In contrast, the manganese analogs [Mn(II) (salophene)] (5) and [Mn(III) (salophene)OAc] (6) inhibited cell growth, caused the programmed cell death only at higher concentrations and did not provoke release of Cu/Zn SOD in any of the four cell lines. Weaker cell death-promoting effects were observed when the salophene moiety of 3 and 4 was replaced with saldach (complexes 7 and 8), indicating the influence exerted by the ligand structure. In conclusion, Schiff base transition metal complexes induce strong inhibitory effects on human lymphoma and leukemia cells.

Anti-HLA-DR Monoclonal Antibody Evokes a Novel Non-Apoptotic Cell Death Pathway In Acute Lymphoblastic Leukemia

AbstractAbstract 3245Whilst modern treatment approaches cure a high number of patients with acute lymphoblastic leukemia (ALL), little progress has been made in the treatment of refractory and relapsed ALL and new treatment approaches are needed. We recently demonstrated that anti-HLA-DR class II monoclonal antibody (mAb) L243 induces a novel non-apoptotic mode of cell death in B-cell lymphoma lines, defined by homotypic adhesion (HA), actin reorganisation and lysosomal activity (Ivanov et al. J Clin Invest, 2009). Here, we extend these important observations and examine whether this novel form of mAb induced cell death occurs in pre-B ALL cell lines.Expression of HLA-DR was determined using flow cytometry in a panel of ALL cell lines (REH, SupB15 and SD1). HLA-DR was expressed at high levels on each of the cell lines. The ability of L243 to induce HA and cell death (Annexin V/PI positivity) was assessed using microscopy and flow cytometry respectively. L243 was able to evoke both strong HA and cell death in all of the ALL cell lines (e.g. in SupB15 cells 46±1.7% death versus 7±0.5% in controls, p<0.001 by Student's t-test). Inhibitors of actin polymerization (cytochalasin D, latrunculin B) were used to assess the role of actin in cell death and HA induced by L243. These inhibitors of actin polymerization inhibited both HA and cell death elicited by L243 (e.g. in SupB15 cells 24±0.5% death versus 10.3±0.8% with Latrunculin B, p<0.001), demonstrating the dependence of HA and cell death on actin reorganisation. The importance of cell to cell contact in this form of antibody induced cell death was confirmed by the addition of low-melting point agarose which physically blocked cell to cell contact and markedly attenuated cell death induced by L243. In contrast using the pan-caspase inhibitor QVD OPH had no effect on cell death induced by L243, indicating that this mode of death is non-apoptotic.These findings demonstrate that anti-HLA DR mAb L243 induces a novel model of cell death in ALL cell lines that is independent of caspase activation and dependent on actin reorganization. This data suggests that this novel mAb induced death pathway is independent of apoptosis and potentially exploitable in the clinic in leukemias resistant to chemotherapy apoptosis induction. Disclosures:No relevant conflicts of interest to declare.

Inhibition of Akt Increases the Sensitivity of Acute Lymphoblastic Leukemia (ALL) to the Glycolytic Inhibitor 2-Deoxy-D-Glucose (2-DG)

AbstractAbstract 3254Cancer cells exhibit an increased dependency on the glycolytic pathway as the predominant energy source over mitochondrial oxidative phosphorylation (OXPHOS). This unique alteration in glucose metabolism gives malignant cells significant proliferative advantages by enabling cancer cells to better adapt to the hypoxic microenvironment. The glucose analogue 2-Deoxy-D-Glucose (2-DG) has been found to be an effective antitumor agent in both animal models and human clinical trials. In childhood acute lymphoblastic leukemia (ALL), elevated glycolytic rates have been identified and increased glucose consumption has been postulated to be responsible for chemotherapy resistance. We have previously reported that B-precursor ALL (Bp-ALL) cells exhibit significant sensitivity to 2-DG under normoxia, and that PTEN mutant T-ALL CCRF-CEM cells are less sensitive to 2-DG compared to Bp-ALL cells. In this study, we further investigated signaling alterations in critical metabolic pathways linked to cell survival and proliferation and the mechanisms of apoptotic cell death following inhibition of glycolysis in ALL. Warburg suggested that the deficiency of mitochondrial respiration plays an important role in the metabolic shift seen in cancer cells. We assessed mitochondrial function in selective Bp- and T-ALL cell models including CCRF-CEM, NALM6, REH (RUNX/ETV1 + Bp-ALL), SupB15 and TOM1 (both BCR/ABL + Bp-ALL). The K562 cell line (CML) and an EBV-immortalized non-malignant lymphocyte cell line HCC1187BL were tested as controls. Our data indicate that oxygen consumption rates, and steady-state levels of representative protein markers of OXPHOS were decreased in the ALL cell lines, which correlated with a decrease in mtDNA levels when compared to CML and EBV immortalized lymphocyte controls. We previously demonstrated that 2-DG also interferes with protein synthesis and processing in ALL cells by inhibiting N-linked glycosylation. On this basis, we investigated the induction of prolonged ER stress leading to an unfolded protein response (UPR) in ALL cells following treatment with 2-DG. The expression of UPR specific markers Grp78/BiP and Grp94 were probed in CCRF-CEM, NALM6, and SupB15 cells using specific antibodies by Western immunoblotting. In the presence of 4mM 2-DG, significant increase in the expression of both Grp78/BiP and Grp94 was detected in all ALL cell lines tested. Western immunoblotting also identified increased expression of CHOP/GADD153 and cleavage of Poly (ADP-ribose) polymerase (PARP) following 2-DG treatment, indicating that 2-DG leads to UPR-induced apoptotic cell death in ALL cells. Among ALL cells studied, the PTEN mutant CCRF-CEM cells exhibit constitutive activation of Akt and resistance to 2-DG. We have reported that simultaneous inhibition of glycolysis and Akt signaling results in greater induction of cell death. In this study we further investigated signaling changes within these pathways to determine the mechanism of synergistic cell death following combination treatment. Western immunoblotting demonstrated that the combination of 2-DG and the Akt inhibitor × (AIX) led to almost complete abrogation P-Akt expression at both Ser473 and Thr308, and significant down-regulation of P-mTOR signaling as compared to treatment with each drug alone. Most important, the combination treatment led to a significant decrease in the expression of Hexokinase and GLUT1 in CCRF-CEM cells, and “sensitized” these cells to apoptotic death by 2-DG as demonstrated by significant cleavage of PARP.These data demonstrate for the first time that ALL cells are unable to effectively utilize mitochondrial OXPHOS for ATP generation, providing an explanation for their sensitivity to 2-DG under normoxic conditions. We also demonstrate that the cytotoxicity of 2-DG in ALL cells is due to concomitant inhibition of N-linked glycosylation leading to ER stress and UPR-induced apoptosis. Finally, our data indicate that hyper-activation of Akt signaling is responsible for the relative resistance of PTEN mutant CCRF-CEM cells to inhibition of glycolysis, and that simultaneous inhibition of Akt signaling is capable of overcoming this relative resistance to 2-DG. We propose that glycolytic inhibitors alone or in combination with selected targeted agents aimed at key metabolic and oncogenic pathways show promise as novel strategies for ALL therapy. Disclosures:No relevant conflicts of interest to declare.

High Pro-Apoptotic Effects of 17-Allylamino-17-Demethoxy Geldanamycin (17-AAG) In Philadelphia Acute Lymphoblastic Leukemia (Ph+ ALL)

AbstractAbstract 3226Heat Shock Protein 90 (HSP 90), a 90 Kda molecular weight protein, is one of the most abundant cytosolic chaperone, acting in protein folding, refolding and degradation and of particular importance in cell survival. HSP 90 is overexpressed in acute leukemia cells and this high expression is related with a poor prognosis. This ATP-dependent chaperone could be an interesting potential drug target. 17-AAG, a Geldanamycin derivates, is a HSP 90 inhibitor with a promising antitumor activity.Very few reports have evaluated the effects of 17-AAG in acute lymphoblastic leukemia (ALL). The aim of this work is to study the cytotoxicity of 17-AAG and to compare Philadelphia positive (Ph+) ALL to common B-cell ALL.Two human leukemia cell lines, Reh (common B-cell ALL) and SUPB15 (Ph+ ALL) were used in this study. We identified 63 consecutive patients treated in our institution for ALL (44 common B-cell ALL and 19 Ph+ ALL) and leukemic samples were collected at diagnosis from bone marrow aspiration after patient's consent.The expression of HSP 90, pro-apoptotic BAX protein, anti-apoptotic bcl-2 and bcl-xl proteins was studied by flow cytometry. Cell lines and ALL patient's cells were cultured in RPMI 1640 and exposed to various concentrations of 17-AAG. Apoptosis was evaluated by an Annexin V and an activated caspase-3 staining by flow cytometry. Results were expressed as percentage of positive cells.Pro-apoptotic effect of 17-AAG in Ph+ ALL cells was superior when compared to common B-cells with a 100% mortality rate after exposure to [10μM] 17-AAG for 24 hours for SUPB15 cell line whereas 24% of cell surviving was noted for Reh cell line. Similar results were observed with patient's leukemic samples (as shown in figure). The susceptibility of Ph+ ALL cells to 17-AAG was confirmed by the assessment of the IC50, estimated at 1.1 μM for SUPB15 cells versus 2.9 μM for ReH cells. [Display omitted] As assessed by Annexin V binding and activated caspase-3 staining, 17-AAG induced apoptosis in ALL cells. As regard Ph+ ALL, the median percentage of Annexin-V positive cells and caspase-3 positive cells after exposure to [5μM] 17 AAG for 24 hours was 54% and 57% respectively and increased up to 99% and 99,5% after exposure for 48 hours. Spontaneous apoptosis in control cell culture was measured in 1% at 24 hours and 3% at 48 hours.The pattern of expression of HSP90 and apoptotic proteins was different between common B-cell ALL and Ph+ ALL cells. The percentage of HSP90 positive cells was 62% for Reh cell line whereas it reached 100% for SUP B15 cell line. As shown in the Table, the percentage of HSP90-positive cells and anti-apoptotic bcl-2 and bcl-xl proteins expression were higher for Ph+ ALL samples when compared to common B-cell ALL samples.HSP90Bcl-2Bcl-xlBaxCommon B-cell ALL samples n=4432.5% (±19.8)33% (± 21.4)28.5% (±19.5)33% (±17)Ph+ ALL samples n = 1979% (±7.5)82% (±8.4)84% (±7.8)12% (±4.3)When ALL cells were exposed in culture to various concentrations of 17-AAG, there was no change in HSP90 expression but we observed a down-regulation in bcl-2 and bcl-xl expression and an up-regulation in bax expression.In summary, we showed that HSP90 and anti-apoptotic proteins were expressed at a higher level on Ph+ ALL cells when compared to common B-cell ALL. High percentage of HSP90-positive cells was associated with high sensitivity to 17-AAG. 17-AAG is a new targeted therapy that induces the apoptotic death of leukemic cells via a caspase-3 dependant way. It would be interesting to test its antileukemic activity in combination with chemotherapeutic agents to study additional or synergistic effects. Despite therapeutic improvement with the development of tyrosine kinase inhibitors (TKI) in the treatment of Ph+ ALL, relapse remains a major problem. Considering that Bcr-Abl constitutes HSP 90 substrates and depends on this chaperone for its maturation and conformational maintenance, 17-AAG could be of particular interest for Ph+ ALL disease, in combination with TKI. We can hypothesise that this drug could restore the sensitivity to TKI treatment for patients with Bcr-Abl mutation. Disclosures:No relevant conflicts of interest to declare.

To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

AbstractAbstract 2135 ObjectiveTo establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. MethodsPh(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. ResultsWe established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. ConclusionIn vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures:No relevant conflicts of interest to declare.

VE-cadherin and PECAM-1 enhance ALL migration across brain microvascular endothelial cell monolayers

Infiltration of the central nervous system (CNS) by leukemia is a problematic disease manifestation of acute lymphoblastic leukemia (ALL). The mechanisms by which leukocytes interact with human brain-derived microvasculature endothelial cells (HBMEC) and enter the CNS are largely derived from models of inflammation. However, our data indicate that ALL cells do not elicit an inflammatory phenotype by HBMEC. Our current investigation focuses on the contribution of the unique coexpression of vascular endothelial (VE)-cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) by ALL in mediating leukemic cell interactions with HBMEC as an in vitro model of the blood-brain barrier. Primary ALL and ALL cell lines were evaluated for VE-cadherin and PECAM-1 expression. Lentiviral-mediated transduction of VE-cadherin and PECAM-1 into REH cells and antibody neutralization of VE-cadherin and PECAM-1 in SUP-B15 cells was used to delineate the role of these two proteins in mediating ALL adhesion to, and migration through, HBMEC monolayers. Although cell line models indicate that VE-cadherin and PECAM-1 expression is found on the surface Philadelphia chromosome-positive ALL, evaluation of primary ALL demonstrates that VE-cadherin and PECAM-1 are expressed independent of Philadelphia status. Expression of VE-cadherin and PECAM-1 by ALL enhanced the adhesion of ALL to HBMEC, while expression of PECAM-1 enhanced ALL adhesion to, and migration through, HBMEC. Expression of VE-cadherin and PECAM-1 by ALL cells positions them to interact with HBMEC. By increasing our understanding of molecular mechanisms through which ALL cells gain entry into the CNS, new strategies may be designed to prevent leukemia cell entry into the CNS.

Abstract 1611: Identification of target molecules and anti-leukemic effects of novel phospha sugar derivative, TMPP

Abstract [Background and Aims] We synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. [Methods] In human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. [Results] TMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. [Conclusion] We demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers. 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 1611.

Mechanisms of Sensitivity of B-Precursor, T-Lineage and BCR/ABL Positive Acute Lymphoblastic Leukemia (ALL) to the Glycolytic Inhibitor 2-Deoxy-D-Glucose (2-DG).

Abstract 3081Poster Board III-18Novel treatment strategies are needed for patients with ALL diagnosed with resistant phenotypes or after relapse. The glucose analogue 2-deoxy-D-glucose (2-DG) is a glycolytic inhibitor that induces growth arrest and cell death by inhibiting the key glycolytic enzymes phosphoglucose isomerase (PGI) and hexokinase (HK). Cancer cells and hypoxic cells are more sensitive to 2-DG due to their reliance on glycolysis for ATP generation. In this study, we evaluated the antileukemic activity of 2-DG in T- and Bp-ALL subtypes characterized by non-random translocations. The cytotoxicity of 2-DG was determined on CCRF-CEM (PTEN mutant T-ALL), NALM6 (Bp-ALL), REH (TEL/AML1+ Bp-ALL), and the BCR/ABL+ ALL cell lines SupB15 and TOM1. Cells were treated with 4mM 2-DG for 72 h under normoxic vs. hypoxic (0.5% O2) conditions using a hypoxia box chamber (BioSpherix), and growth arrest and cell death were assessed. 2-DG induced 2-13 fold higher cell death in all ALL cells tested under normal O2 conditions compared to hypoxia. Similarly, growth inhibition was greater under normoxia. Both BCR/ABL+ ALL cell lines TOM1 and SupB15 were among the most sensitive, while the PTEN mutant CCRF-CEM (T-ALL) cell model was the least. To analyze the mechanisms of higher sensitivity of ALL cells under normoxia, we first used mannose to assess the role of glycosylation in 2-DG induced ALL cell death. Mannose reversed 2-DG induced cell death in CCRF-CEM cells but only partially in NALM6 cells, indicating inhibition of glycosylation mainly mediated death in CCRF-CEM cells, while inhibition of both glycolysis and glycosylation mediated cell death in NALM6 cells. To further assess other mechanisms leading to cell death in ALL, we evaluated mitochondrial integrity in representative Bp- and T-ALL models by determining cellular respiration with a Clark electrode (Hansatech Instruments). Our data show that cellular respiration in NALM6 was 50% lower compared to CCRF-CEM cells, suggesting that an intrinsic mitochondrial dysfunction was also responsible for 2-DG induced cell death in NALM6 cells. We then evaluated the observed higher sensitivity of BCR/ABL+ ALL cells to 2-DG. The AMPK and PI3K/Akt/mTOR pathways regulate protein, fatty acid and glucose metabolism, and key glycolytic enzymes are known to be upregulated in BCR/ABL+ cells. Therefore, we determined changes on AMPK and PI3K/Akt/mTOR signaling following exposure to 2-DG in SupB15 (BCR/ABL+) and in CCRF-CEM cells, the least sensitive ALL model examined. Western immunoblotting showed that 2-DG led to upregulation of p-AMPK (Thr172) and downregulation of p-mTOR (S2448) and p-p70S6K (Thr389). In both cell lines, 2-DG also led to cell death by triggering an unfolded protein response (UPR) evidenced by CHOP expression and PARP cleavage. We previously demonstrated that activation of AMPK leads to upregulation of Akt as a compensatory survival mechanism in ALL cells (Mol Cancer 6: 46, 2007). On this basis, we then tested induction of cell death by the combination of 2-DG (4 mM) plus Akt inhibitor X (AIX) (12 μM) in CCRF-CEM vs. SupB15 cells under normoxic conditions. Again, SupB15 cells were more sensitive to 2-DG, while AIX alone at low dose (12 μM) had minimal effect on either cell line. When both drugs were used in combination at these same concentrations, significant synergism was seen in CCRF-CEM cells but not in SupB15 cells. Analysis of AMPK, mTOR and Akt in CCRF-CEM cells treated with the combination of 2-DG plus AIX showed higher p-AMPK activation and mTOR downregulation compared to 2-DG alone, while these signaling changes were lower in SupB15 cells. Consistent with these findings, this combination led to higher induction of UPR and PARP cleavage in CCRF-CEM vs. SupB15 cells. We conclude that the greater sensitivity of ALL cells to 2-DG under normoxia is due to concomitant inhibition of glycolysis and/or glycosylation, an inherent mitochondrial dysfunction in some phenotypes, and 2-DG induced changes in AMPK and PI3K/Akt/mTOR signaling leading to the induction of UPR. Concomitant Akt inhibition sensitizes the relatively 2-DG resistant CCRF-CEM cells to death with 2-DG. Chemotherapy resistant BCR/ABL+ ALL cells exhibit significant sensitivity to 2-DG, likely due to their increased reliance on glycolysis for ATP generation. On this basis, we propose that glycolytic inhibitors represent a promising novel therapeutic strategy for ALL, particularly for BCR/ABL+ ALL. DisclosuresNo relevant conflicts of interest to declare.

To Study the Proliferative Inhibition of Anticancer Drug in Two Kinds of Ph (+) Leukemia Cell Lines.

Abstract 4110 ObjectiveTo study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods(1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results(1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion(1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures:No relevant conflicts of interest to declare.

Identification of Target Molecules and Anti-Leukemic Effects of Novel Phospha Sugar Derivative, TMPP.

Abstract 4814 Background and AimsWe synthesized two phospha sugar derivatives, 2,3,4-tribromo-3-methyl-1-phenylphospholane 1-oxide (TMPP) by reacting 3-methyl-1-phenyl-2-phospholene 1-oxide with bromine, and we has reported their potential as an antileukemic agent in cell lines. In this study, we have investigated that TMPP have antileukemic effects as shown in various leukemia cell lines as well as primary AML patient specimens. Moreover, we have identified the target molecules of TMPP by using docking simulation. MethodsIn human leukemia cell lines (HL60, NB4, U937, NOMO-1, CEM, MOLT4, SUP-B15, and YRK2 cells), the anti-leukemic effects were elevated by cell proliferation assay, Cell cycle analysis, Western blotting. In AML cells derived from AML patients and normal hematopoietic progenitor cells derived from healthy volunteers, the changes of viability by treatment with TMPP were evaluated. By using docking simulation, the target molecules were identified in leukemia cells treated with TMPP. ResultsTMPP showed inhibitory effects on leukemia cell proliferation, with mean IC50 values of 10.2 nmol/L for TMPP, indicating that inhibition appeared to be dependent on the number of bromine atoms in the structure. Further, TMPP at 8.4 nmol/L induced G2/M cell cycle block in leukemia cells, and TMPP at 17.3 nmol/L induced apoptosis in these cells. TMPP treatment reduced cell cycle progression signals (FoxM1, KIS, Cdc25B, Cyclin D1, Cyclin A, and Aurora-B) and tumor cell survival (Bcl-2, p27Kip1 and p21Cip1). Further, treatment with TMPP significantly reduced the viability of AML specimens derived from AML patients, but only slightly reduced the viability of normal ALDHhi progenitor cells. We also found that TMPP showed the strong activity against Aurora kinase B and Bcl-2 by using docking simulation. ConclusionWe demonstrate that TMPP inhibits Aurora kinase B and Bcl-2 and shows a dominant Aurora B kinase inhibition-related cell cycle arrest and the induction of mitochondrial potential catastrophe-related apoptosis in acute leukemia cells. Moreover, we identified Aurora kinase B and Bcl-2 as the target molecules of TMPP. We conclude that TMPP has great therapeutic potential in anticancer therapy in a wide range of cancers. Disclosures:No relevant conflicts of interest to declare.