pre-B Acute Lymphoblastic Leukemia Cell Lines Research Articles

A Direct Comparison of the In Vivo Efficacy and in Vitro Signaling of Chimeric Antigen Receptors and Endogenous T Cell Receptors

The adoptive transfer of chimeric antigen receptor (CAR) expressing T cells targeting the CD19 antigen induces remission in 70-90% of pediatric patients with relapsed and/or refractory acute lymphoblastic leukemia (ALL). Despite initial enthusiasm, emerging data indicate the durability of remission is limited in a substantial number of patients, therefore, addressing relapse will be critical for increasing the clinical efficacy of CAR T cell therapy. CARs are synthetic receptors designed to redirect the antigen specificity of T cells while recapitulating the T cell activating properties of the endogenous T cell receptor (TCR). Clinical and pre-clinical observations have demonstrated that CAR T cells have inferior sensitivity to low levels of antigen and have diminished persistence in vivo compared to T cells responding to TCR activation. These observations suggest the CAR does not fully replicate the signaling of a TCR which subsequently leads to suboptimal T cell function which may underlie the inability of CAR T cell therapy to induce durable remissions in a significant number of patients.In these studies, we directly compared the in vivo and in vitro consequences of CAR versus TCR activation, as well as the signal transduction of each of these receptors. To accomplish this, we created a novel, syngeneic model system in which a 2nd generation CAR is introduced into OTI TCR transgenic T cells to generate a population of T cells (CAR-OTI cells) in which each cell recognizes the CD19 antigen via its CAR as well as the ovalbumin (OVA) antigen via its TCR, thus providing a model in which the function and signaling of each receptor could be studied within a uniform population of T cells. To stimulate either the CAR or the TCR, we generated antigen-expressing cell lines from the pre-B ALL cell line, E2aPbx, that expressed either the cognate antigen for the CAR (CD19), the TCR (OVA) or both. These cell lines allowed us to stimulate either receptor in a model which preserved the properties of the cell-to-cell interactions expected in vivo . We validated the activity of each receptor in this model, by demonstrating similar in vitro degranulation, proliferation and target cell lysis in CAR-OTI cells upon co-incubation with CAR-antigen or TCR-antigen positive E2aPbx cells. Despite the similarities seen in vitro, CAR-OTI cells adoptively transferred into mice with established E2aPbx leukemia demonstrated enhanced expansion at 4 (p<0.001) and 6 days (p<0.01) post-infusion in TCR-activated CAR-OTI cells, relative to CAR-activated CAR-OTI cells. Likewise, clearance of leukemia from the bone marrow (p<0.001) and survival (p<0.01) were each superior in mice in which CAR-OTI cells were activated through the TCR, relative to the CAR. These results demonstrate the significant difference in in vivo behavior and efficacy of CAR- versus TCR-mediated T cell activation, and suggest these differences may result from differences in the signals generated by each receptor.Using our model of CAR-OTI cells and E2aPbx cells expressing either the CAR- or TCR-antigen, we interrogated proteins in the canonical TCR signal transduction cascade after CAR or TCR activation. We demonstrated that CAR activation resulted in increased and prolonged phosphorylation of Zap70, a kinase central to canonical TCR signal transduction. Despite the increased activation of Zap70, the CAR signal demonstrated diminished activation of the MAPK family members, Erk (p<0.001) and p38 (p<0.05), which correlated with the diminished in vivo efficacy seen in this model. Furthermore, this model was used to define the global phosphoproteome of CAR- and TCR-activated CAR-OTI cells by a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) methodology in conjunction with mass spectrometry (MS). Our SILAC-MS analysis demonstrated multiple differences in the signal transduction cascade of CARs relative to TCRs, including proteins of the signalosome as well as the NFAT and NF-kB pathways. These data demonstrate that a CAR generates an “altered” signal, relative to that of a TCR, correlating with diminished in vivo expansion and efficacy in the treatment of established leukemia. These findings underscore the need to better understand the biology of chimeric antigen receptors as a means of rationally altering their design to improve their clinical effectiveness in mediating durable remissions in patients with relapsed/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.

Recurrent NR3C1 Aberrations at First Diagnosis Relate to Steroid Resistance in Pediatric T-Cell Acute Lymphoblastic Leukemia Patients.

The glucocorticoid receptor NR3C1 is essential for steroid-induced apoptosis, and deletions of this gene have been recurrently identified at disease relapse for acute lymphoblastic leukemia (ALL) patients. Here, we demonstrate that recurrent NR3C1 inactivating aberrations—including deletions, missense, and nonsense mutations—are identified in 7% of pediatric T-cell ALL patients at diagnosis. These aberrations are frequently present in early thymic progenitor-ALL patients and relate to steroid resistance. Functional modeling of NR3C1 aberrations in pre-B ALL and T-cell ALL cell lines demonstrate that aberrations decreasing NR3C1 expression are important contributors to steroid resistance at disease diagnosis. Relative NR3C1 messenger RNA expression in primary diagnostic patient samples, however, does not correlate with steroid response.

Suppression of c-Myc using 10058-F4 exerts caspase-3-dependent apoptosis and intensifies the antileukemic effect of vincristine in pre-B acute lymphoblastic leukemia cells.

Despite an old history behind the identification of the leading role of c-Myc in leukemogenesis, the road to constructing a therapeutic perspective for this molecule in acute lymphoblastic leukemia (ALL) is yet mesmerizing. This study was designed to provide a better outlook for the anticancer property of 10058-F4, an appealing inhibitor of c-Myc, in pre-B ALL cell lines either in the context of monotherapy or in combination with chemotherapeutic drugs. Our results declared that abrogation of c-Myc decreased the proliferative capacity of pre-B ALL-derived cells through halting the transition of the cells from G1 phase, and reducing the replicative potential of both REH and Nalm-6 cells, at least partly, through c-Myc-mediated suppression of human telomerase reverse transcriptase. Moreover, 10058-F4 potently induced a caspase-3-dependent apoptosis in pre-B ALL cells via shifting the balance between pro- and anti-apoptotic target genes. Although the inhibition of PI3Kδ using Idelalisib upregulated the messenger RNAexpression of autophagy-related genes in 10058-F4-treated cells, treatment with autophagy inhibitor chloroquine decreased viability of the cells, either as a single agent or in combination with Idelalisib and/or 10058-F4; suggesting that the activation of autophagy in pre-B ALL cells could blunt apoptotic events and attenuate anticancer effect of both c-Myc and PI3K inhibitors. Finally, the results of our synergistic experiments delineated that 10058-F4 produced a synergistic effect with vincristine and provided an enhanced therapeutic efficacy in ALL cells, highlighting that c-Myc oncoprotein could be a bona fide target for the treatment of ALL.

CD4 CAR T Cells Mediate CD8-like Cytotoxic Anti-Leukemic Effects Resulting in Leukemic Clearance and Are Less Susceptible to Attenuation By Endogenous TCR Activation Than CD8 CAR T Cells

Adoptive immunotherapy using T cells armed with chimeric antigen receptors (CAR) has proven extremely effective against CD19+ B-lineage acute lymphoblastic leukemia (ALL) with remission rates as high as 70-90% in recent clinical trials in relapsed/refractory patients. CD8 T cells are typically thought of as the primary antitumor effector cells in an adoptively transferred product due to their potent cytolytic capabilities, whereas CD4 T cells are thought to primarily provide "help" to enhance CD8 T cell activity via cytokine production. However, CARs are synthetic constructs that likely alter the functionality of T cells. Further, CAR T cells are endowed with 2 specificities, one through the CAR and one through the endogenous T cell receptor (TCR). To evaluate the biology of CAR T cells, we sought to evaluate the impact of CAR expression on the functionality of T cells and to study the impact of TCR on CAR T cell activity using both human CAR T cells in a xenograft model and murine CAR T cells in a syngeneic murine model. A human or murine second-generation anti-CD19 scfv/CD28/CD3ζ CAR was transduced into human or mouse CD8 (CAR8) and CD4 (CAR4) T cells, respectively, and tested against pre-B ALL cell lines in human murine xenografts or syngeneic models. Surprisingly, human CAR4 cells alone had equivalent ability to eradicate the Nalm6 ALL in vivo as CAR8 cells. Although CAR8 cells more rapidly cleared leukemia (2 vs 4 days), relapse eventually occurred. In contrast, CAR4 cells eradicated leukemia more slowly, but persisted longer and prevented relapse. In mice receiving CAR4+CAR8 cell products, only CAR4 cells were detectable at day 55. Next we utilized our syngeneic murine system to study CAR4 and CAR8 activity in an immunocompetent system using TCR transgenic T cells with known TCR specificity against the male histocompatibility antigen, HY. As expected, when stimulated through the TCR, CAR8 cells produced cytokines and degranulated, as manifested by CD107a expression, whereas CAR4 cells only demonstrated a cytokine production response. However, murine CAR4 cells activated through the CAR receptor develop "CD8-like" cytolytic features characterized by degranulation and were able to completely eradicate leukemia in vivo when administered without CAR8 cells, similar to the human CAR4 cells. We then evaluated the effect of TCR antigen in vivo on CAR T cell function by comparing activity in male (HY+) vs female (HY-) recipients. CAR4 cells were curative in both male and female recipients. However, CAR8 treatment has no anti-tumor activity in male recipients indicating that CAR8 cells are very susceptible to the negative effects of TCR signaling. TCR antigen availability in vivo greatly increased the number of infused CAR4 cells (P=0.003, male vs female recipients), while they significantly decreased the number of CAR8 cells (P=0.0023, male vs female recipients) after 1 week. Interestingly, we found that there was significant down-regulation of CAR expression on both CAR4 and CAR8 cells in male recipients (HY+). PD-1 and Tim3 were expressed at low levels on CAR4 cells and CAR8 cells in female recipients. However, there was significantly higher PD-1 (P=0.0079) and TIM3 (p=0.014) expression on CAR8 cells in male vs female recipients, whereas there was no difference in PD-1 or Tim3 on CAR4 cells recovered from male vs female recipients. These data indicate that CAR4 cells are more functional and become less exhausted than CAR8 cells in the presence of TCR antigen. Finally, we evaluated the long-term persistence of CAR4 and CAR8 cells in our syngeneic model in which there is no xenogeneic reactivity allowing for long-term monitoring. At day 60 and day 80, CAR4 cells were detectable in bone marrow and spleen, whereas CAR8 cells had completely disappeared. Despite the long-term persistence of CAR4 cells in recipients with or without TCR antigen, CAR4 cells are not completely immune to effects of TCR activation. In male recipients after 60 days, CAR4 cells also start to express high PD-1 levels and have decreased CAR T cell counts. These findings have important implications for CAR therapy, particularly in the allogeneic setting where the presence of TCR antigen may be more likely to be present in the recipient. In addition, these results suggest that CAR4 cells alone are cytolytic, equally potent to CAR8 cells at eradicating leukemia in vivo, and may be superior due to better persistence and reduced susceptibility to exhaustion. DisclosuresNo relevant conflicts of interest to declare.

Immunotherapeutic Targeting of EphA3

Eph receptors form the largest family of receptor tyrosine kinases. Eph receptors interact with cell-surface ephrin ligands to direct cell growth and migration. High expression of Eph receptors in a number of cancers has been linked to progression through facilitation of invasiveness and metastatic spread. EphA3 protein, which was originally described in leukemia, has also reported to be expressed in sarcomas, lung cancer, prostate cancer, melanoma and glioblastoma.The EphA3-specific monoclonal antibody, IIIA4, binds and activates human and mouse EphA3 with similar affinities. Binding is followed by internalization of receptor-antibody complexes. High expression of EphA3 has been reported on LK63 pre-B acute lymphoblastic leukemia (ALL) cell line and contrasts with lack of expression of EphA3 in the Reh, a similar pre-B ALL cell line. We investigated the effect of IIIA4 monoclonal antibody treatment in LK63 and Reh NOD/SCID xenograft models. In the LK63 xenograft model, administration of the IIIA4 antibody led to inhibition of tumor growth and decreased spread from bone marrow to the spleen and other organs with a corresponding increase in survival, however no reduction in engraftment was observed in Reh xenograft model, suggesting that the effect was directed against the leukemic cells rather than the stromal and vascular elements in these tumors.To confirm the tumor-specific effect of IIIA4 therapy, LK63 EphA3-knockdown and Reh EphA3-expressing cell lines were developed and tested in the xenograft model of leukemia. Similar to LK63 xenograft model, upon treatment with IIIA4 monoclonal antibody, the EphA3-expressing Reh xenografts showed reduction in the bone marrow engraftment and slowing of the disease progression. Consistent with the effect of EphA3 monoclonal antibody treatment on Reh xenograft model, the LK63 EphA3-knockdown xenografts showed minimal difference between treated and control group but had a notably significant reduction in the splenic engraftment compared to the normal LK63 model.To understand the mechanism leading to therapeutic effect of IIIA4 on EphA3-expressing leukemic xenografts, the effect of EphA3 activation on the LK63 and LK63 EphA3-knockdown cells was examined. This analysis showed elevated level of Src kinase activity upon stimulation with either IIIA4 or ephrinA5-Fc. Src kinases have been implicated in Eph-mediated signalling in other systems, and its activation appears to target adhesive mechanisms, particularly integrins, and cell motility functions consistent with the observed effects of antibody on cell movement.Given the capacity of the antibody to induce internalisation of EphA3, the possibility of linking a cytotoxic payload to IIIA4 was examined. The alpha-particle emitting radio-active isotope bismuth-213 was linked to IIIA4 and tested in the LK63 model with a considerably enhanced therapeutic effect specifically a significant increase in survival. More importantly, no toxicity to normal tissues was observed, as EphA3 is expressed at very low level or is entirely absent on normal tissues.A Humaneered® form (FA225-C10) of IIIA4 antibody has been generated for clinical development. This antibody binds to EphA3 with an improved affinity and has retained the direct apoptotic activity of mouse IIIA4 antibody. In the xenograft model of leukemia the antibody directly affects the tumor cells with no effect on stromal and vascular elements. In human tumor xenografts of prostate cell line DU-145 in nude mice, FA225-C10 antibody produced a significant inhibition of tumor growth. In this model the tumor cells do not express EphA3 but the tumor stroma and neovasculature express EphA3 and treatment with antibody reduced both tumor stroma and new vessel density.In Summary targeting EphA3 tumor cells and/or tumor stromal fibroblasts may therefore constitute a novel approach to treating solid tumors as well as hematologic malignancies. The Phase 2 component of a clinical study in acute myeloid leukemia (AML), myelodysplastic syndrom (MDS) and myelofibrosis (MF) patients is ongoing in which the activity of KB004 will be characterized. Future clinical studies might be extended to other cancers including acute lymphoblastic leukaemia (ALL) and other solid tumors. DisclosuresYarranton:Glaxo: Equity Ownership; Stemline Therapeutics: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; KaloBios: Employment.

POL5551, a Novel and Potent CXCR4 Antagonist, Enhances Sensitivity to Chemotherapy in an in Vivo Model of High-Risk (HR) Pediatric Acute Lymphoblastic Leukemia (ALL)

Background: Interaction between surface receptor CXCR4 (s-CXCR4) and chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow (BM) microenvironment. We previously demonstrated: 1) chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) and ALL enhances stromal protection from chemotherapy-induced apoptosis; 2) the FDA-approved CXCR4 inhibitor plerixafor reverses stromal protection and chemotherapy resistance both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged (MLL-R) ALL; 3) the novel Protein Epitope Mimetic POL5551, a selective and potent antagonist of CXCR4, blocks the SDF-1-binding site of CXCR4, inhibits SDF-1-induced chemotaxis, and reverses stromal-mediated protection from chemotherapy in vitro in pre-B and T ALL cell lines. Here, we further characterize the effects of POL5551 (POL) on surface adhesion molecule expression in ALL, and its in vivo effects in a xenograft model of HR pediatric ALL.In Vitro Methods/Results: We have previously shown that POL inhibits 12G5 antibody binding to s-CXCR4 in ALL cell lines, suggesting an overlapping of the two binding sites. We sought to verify these results using primary samples of pediatric ALL. We treated 3 pre B and 3 T cell ALL primary samples with a dose range of POL and measured s-CXCR4 by FACS at multiple time points. POL inhibition of 12G5 binding was potent (average IC50 at 2 hours pre B 8.3 nM, T 1.4 nM), rapid (<1 hour) and sustained (>24 hours). Further, POL was significantly more potent at inhibiting 12G5 binding than plerixafor (average IC50 at 2 hours pre B 18.4 nM, T 8.4 nM). To further characterize POL’s effects in ALL, we treated 2 pre B and 2 T cell ALL cell lines with POL or vehicle control and then treated with SDF-1α or vehicle control. Treatment with POL inhibited SDF-1α-induced phosphorylation of ERK1/2 in a dose-dependent manner. In parallel, we measured POL-induced compensatory upregulation of the alternative surface adhesion molecules CXCR7 and VLA-4 (CD49d), and found that POL led to increased CXCR7 expression at early time points that began to decrease after 24 hours. We did not find a consistent effect of POL on CD49d surface expression.Xenograft Methods/Results: Infant MLL-R ALL primary samples (n=4) were transplanted into sublethally irradiated NSG mice. After 2 weeks, mice were treated on days 1-3 of 2 consecutive weeks with 1) vehicle control (C), 2) POL (5 mg/kg SC), 3) AraC (200 mg/kg IP), or 4) POL followed by AraC 4 hours later (POL+AraC). One week after treatment, cells were harvested from BM, spleen, and peripheral blood (PB). Leukemic blasts were defined as human CD19+ and CD45+. Overall leukemic burden (average % blasts in BM+spleen+PB) did not differ between mice treated with either C (56.2%) or POL (49.5%, p=0.12). However, treatment with AraC (36.7%, p=3E-07) or POL+AraC (26.3%, p=4E-15) significantly decreased total leukemic burden compared to C. Notably, POL+AraC significantly decreased total leukemic burden compared to AraC alone (p=0.001), demonstrating that POL increased overall sensitivity to AraC. When analyzed by organ-specific leukemic burden, POL+AraC resulted in decreased leukemic burden compared to AraC alone in BM (42.8 vs. 49.8%, p=0.27), spleen (16.9 vs. 30.8%, p=0.002), and PB (19.3 vs. 29.6%, p=0.008). Interestingly, AraC and POL+AraC led to significantly increased CXCR7 expression (blasts from BM p<0.01, spleen p<0.04, and PB p<0.03 vs. C) as well as a trend toward increased CD49d expression compared to C.Conclusions: POL5551 is a potent antagonist of s-CXCR4 in pediatric ALL cell lines and primary samples. Surface expression of adhesion molecules in ALL blasts is dynamic and affected by anti-leukemic therapy. Importantly, POL5551 enhances sensitivity to AraC in a xenograft model of infant MLL-R ALL, a HR subtype of pediatric ALL. Our findings support the continued development of BM microenvironment-targeted agents as a therapeutic strategy for pediatric ALL. DisclosuresRomagnoli:Polyphor Ltd.: Employment. Douglas:Polyphor Ltd.: Employment. Tuffin:Polyphor Ltd.: Employment. Zimmermann:Polyphor Ltd.: Employment.

Interleukin-15 enhances cellular proliferation and upregulates CNS homing molecules in pre-B acute lymphoblastic leukemia

AbstractGenome-wide association studies have consistently implicated the interleukin-15 (IL-15) gene in acute lymphoblastic leukemia (ALL) biology, including associations with disease susceptibility, and increased risk of central nervous system (CNS) involvement. However, whether pre-B ALL blasts directly respond to IL-15 is unknown. Here, we show that most pre-B ALL primary samples and cell lines express IL-15 and components of its receptor and that primary pre-B ALL cells show increased growth in culture in response to IL-15. Investigation of mechanisms of action using IL-15–responsive SD-1 cells shows this growth advantage is maximal under low-serum conditions, mimicking those found in cerebrospinal fluid. IL-15 also upregulates PSGL-1 and CXCR3, molecules associated with CNS trafficking. Investigation of downstream signaling pathways indicates that IL-15 induces signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinase (ERK) 1/2, and to a lesser extent phosphatidylinositol 3-kinase (PI3K) and nuclear factor κB (NF-κB) phosphorylation. The IL-15–mediated growth advantage is abolished by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK), PI3K, and NF-κB inhibitors but preserved in the presence of STAT5 inhibition. Together, these observations provide a mechanistic link between increased levels of IL-15 expression and leukemogenesis, high-risk disease, and CNS relapse and suggest potential therapeutic targets.

CITED2 Facilitates Apoptosis Induced By Histone Deacetylase Inhibitor SAHA In Human Pediatric Pre-B Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood, representing 31% of all tumors, and about 85% of children with ALL have B-cell ALL. Although the survival rate is approaching 90%, ALL remains the main cause of death from disease in children and young adults. The activity of histone deacetylases (HDAC) in childhood ALL is increased compared with that in normal peripheral blood mononuclear cells or bone marrow cells. Treatment of mice engrafted with T or B-ALL cells with HDAC inhibitor (HDACi) increases the acetylation of Histone 3 and Histone 4 and prolongs survival of these mice. <>Vorinostat (Suberoylamilide Hydroxamic Acid, SAHA) was the first HDACi approved by the FDA for the treatment of refractory cutaneous T-cell lymphoma. Currently, several clinical trials are being conducted to evaluate its effects on other cancers, including ALL. However, some patients are resistant to HDACi therapy, and concerns regarding toxic side effects of HDACi exist due to the roles of HDACs in multiple pathways. Therefore, identification of new therapeutic targets is required which could improve the efficacy of HDACi by reducing the dose of HDACi administered without compromising the treatment benefits but alleviating the side effects of HDACi. <>CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)–rich tail 2 (CITED2) is a cytokine-inducible gene that plays various roles during mouse development and, in particular, is essential for normal hematopoiesis. While the role of CITED2 in the pathogenesis of leukemia is currently unclear, dysregulation of CITED2 has been implicated in various types of leukemia, including ALL, in which downregulation of CITED2 is frequently observed. In this study, we tested the hypothesis that CITED2 may enhance the sensitivity of human pediatric pre-B ALL cells to HDACi SAHA. <>SAHA treatment of NALM-6 and 697 cells (human pediatric pre-B ALL cell lines) significantly induced apoptosis and cell cycle arrest in a dose dependent manner. The protein level of CITED2 was not affected by SAHA treatment. Although overexpression of CITED2 alone only slightly increased apoptosis, it significantly enhanced apoptosis resulted from SAHA treatment in both NALM-6 (15.1% versus 39.2%) and 697 cells (9.4% versus 14.6%) as assessed by annexin V/Propidium Iodide double staining and flow cytometry analysis (Figure 1). Accordingly, compared with control (i.e. NALM-6 cells transduced with GFP), overexpression of CITED2 also greatly reduced mitochondrial membrane potential of NALM-6 cells caused by SAHA treatment. [Display omitted] To explore the potential mechanisms underlying enhanced apoptosis by overexpression of CITED2 in NALM-6 cells treated with SAHA, we determined the levels of pro- and anti- apoptotic proteins by Western blot and real-time quantitative PCR. We found that SAHA treatment increased the levels of pro-apoptotic molecules Bak, Puma, and Noxa, and decreased the levels of anti-apoptotic molecule Bcl-xL and apoptosis inhibitors XIAP and survivin. Importantly, overexpression of CITED2 markedly increased the protein levels of pro-apoptotic molecules Bak and Bim. Furthermore, knockdown of Bim by shRNA significantly attenuated apoptosis in Cited2 overexpressing NALM-6 cells treated with SAHA.Taken together, these results suggest that modulation of the CITED2 activity may confer its cooperative effect with SAHA in pre-B ALL cells and warrant future evaluation of such a combination in inducing apoptosis of primary ALL cells. Disclosures:No relevant conflicts of interest to declare.

The Role of MXD3 in Human Precursor B Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3523Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies.MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls.Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures:No relevant conflicts of interest to declare.

Efficacy of a CD22-targeted antibody-saporin conjugate in a xenograft model of precursor-B cell acute lymphoblastic leukemia

Targeted therapies, such as those using imatinib and rituximab, have revolutionized the treatment of Philadelphia chromosome-positive and CD20-positive acute lymphoblastic leukemia (ALL) respectively, yet these therapies are effective in only a subset of patients and remission is generally not durable. The next generation of targeted therapies includes the use of antibodies conjugated to potent cytotoxic agents and are classified as antibody drug conjugates (ADC). For B-lineage ALL, CD22 is an ideal target for ADC therapy because it is expressed on the majority of B-lineage ALL cells and because antibody binding mediates receptor internalization. HB22.7-SAP is a conjugate of our anti-CD22 monoclonal antibody (mAb), HB22.7, and the ribosome inhibiting protein, saporin (SAP). In vitro, HB22.7-SAP effectively bound to CD22 on the surface of pre-B ALL cell lines and exhibited potent and specific cytotoxicity. In a NOD/SCID xenograft mouse model of pre-B ALL, when compared to the vehicle-treated control, HB22.7-SAP increased the median survival time from 20 days to over 50 days without significant toxicity.

Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Abstract 754 Introduction:Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods:A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results:Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion:Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures:No relevant conflicts of interest to declare.

Targeting AKT Signaling in Pediatric Acute Lymphoblastic Leukemia with Sulforaphane

Abstract 1521Refractory and relapse acute lymphoblastic leukemia (ALL) is one of the leading causes of cancer-related death in children. Novel agents are still in need as a frontline therapy in high risk patients and salvage agent in relapse patients. Leukemic cells acquire a survival advantage over normal blood cells by losing control of the cell cycle and activating pro-survival signals. There are emerging evidences showing that the PI3K/AKT/mTOR pathway is frequently activated in both T cell acute lymphoblastic leukemia (T-ALL) and precursor B cell acute lymphoblastic leukemia (pre-B ALL) (approximately 30% of ALL patients). Thus, drugs able to inhibit AKT signaling are potential candidates for ALL treatment. Sulforaphane (SF) is a dietary compound found in cruciferous vegetables with well-known chemopreventive and chemotherapeutic function in solid tumors by inducing apoptosis and inhibiting survival and cell division. However, the anti-leukemic capacity of SF in hematologic malignancies has not been extensively investigated. In this study, we found increased cleavage of PARP, caspase-3, caspase-8 and caspase-9 by immunoblots in pre-B and T-ALL cell lines treated with SF (5–10 μM) for 24 –48 hours. In addition, we detected increased expression of p21 and cyclin B that correlated with a G2/M block and DNA damage observed in SF treated cells by analysis of DNA content and H2AXγ expression. Noteworthy, SF-treated pre-B and T-ALL cell lines exhibit lower levels of total and phosphorylated AKT in addition to decreased levels of total and phosphorylated mTOR, which was independent of p53 and PTEN activities. Lower survival signals upon SF treatment correlated with a dose- and time-dependent cytotoxicity in pre-B ALL cell lines (Nalm-6, REH and RS-4) and T-ALL cell lines (Jurkat, RPMI, DND-41 and KOPTK1) compared to EBV-transformed lymphoblasts used as a control. Interestingly, SF showed a synergistic effect with vincristine in pre-B and T-ALL cell lines, suggesting a potential use of SF in adjunctive therapy. Aiming to support future clinical trials, we confirmed that SF is effective in lymphoblasts from pediatric patients suffering with ALL, both pre-B and T, in a time- and dose-dependent manner (95% confidence interval IC50: pre-B ALL = 9.9 – 12.13 μM, T-ALL = 8.3 –11.00 μM). To investigate whether SF is effective in vivo in a xenograft mouse model, we injected nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with Nalm-6 cells transduced with GFP-FFluc for bioluminescent imaging detection. SF treatment (2 mg i.p. daily) significantly reduced tumor burden compared to the control group (injected with vehicle). Collectively, our data show that SF has anti-leukemic properties in ALL, both in primary samples from patients and cell lines, by inducing cell death selectively in leukemic cells likely by inhibiting AKT-dependent survival signals. Disclosures:No relevant conflicts of interest to declare.

Para-NO-aspirin inhibits NF-κB and induces apoptosis in B-cell progenitor acute lymphoblastic leukemia

Although patients with acute lymphoblastic leukemia (ALL) usually achieve complete remission, disease relapse is common and difficult to treat. Para-NO-aspirin (para-NO-ASA) is a novel drug with demonstrated efficacy against a number of solid tumors and most recently chronic lymphocytic leukemia. In this study, we used ALL cell lines to assess the effects on cell viability by flow cytometry and investigated the mechanism of cell death using chemical inhibitors of key molecules and assessed the effects by flow cytometry, electrophoretic mobility shift assay, Western blotting, and quantitative reverse transcription polymerase chain reaction. Para-NO-ASA induced cell death in the pre-B ALL cell lines in association with increased reactive oxygen species, and suppression of nuclear factor-κB (NF-κB) activity. Chemical inhibitors of NF-κB similarly induced apoptosis in ALL cells, suggesting a role for suppression of NF-κB in para-NO-ASA-induced cell death. Modulation of NF-κB was not via regulation of IκB but potentially through suppression of ROCK1 and loss of reduced glutathione. Our results demonstrate that para-NO-ASA potently induces apoptosis in B-lineage ALL cells via a reactive oxygen species-dependent mechanism that is associated with suppression of NF-κB activity.

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

VPREB1 Deletions Occur Independent of Lambda-Light Chain Rearrangement and Predict Worse Outcome In Pediatric Acute Lymphoblastic Leukemia (ALL)

AbstractAbstract 273VPREB1 deletions occur in 1/3 of pediatric ALL (pALL) patients, but have been considered a normal consequence of λ light chain rearrangement due to VPREB1‘s location among λ chain variable region genes. Recently, VPREB1 deletions were reported in high risk pALL samples with BCR-ABL1-like gene expression (Den Boer 2009) and VPREB1 under-expression was found in a 38-gene signature associated with relapse (Kang 2010). We characterized VPREB1 deletions to determine if they are part of normal λ light chain rearrangement or represent a bona fide copy number alteration (CNA) that predicts worse outcome in pALL. We first used the genome-wide Molecular Inversion Probe (MIP) 330K Cancer Panel (Affymetrix) to identify VPREB1 deletions in a cohort of 52 pALL patients from the University of Utah (n=47 Precursor B-cell (Pre-B), 4 Precursor T-cell (Pre-T), 1 relapse Pre-B ALL). We found focal VPREB1 deletions in 25% (n=12/48) of Pre-B ALL and no (n=0/4) Pre-T ALL patients. We also identified a distinct and often homozygous deletion (14 kb in length) located ≂f 78 kb upstream of VPREB1 that could represent a potential enhancer region; this possible enhancer deletion was seen in 16.7% (n=8/48) of Pre-B and none of the Pre-T ALL patients. 50% of Pre-B ALL samples had at least one deletion of B-cell developmental gene (SPI1, IKZF1, BCL11A, TCF3, EBF1, PAX5, FOXP1, LEF1, VPREB1, BLNK), and of these, half (n=12/24) had deletion only of VPREB1 or its possible enhancer. We next sorted blood leukocytes from 10 healthy volunteers for quantitative PCR (qPCR) analysis on κ V-J junction (deletion=attempted κ rearrangement), λ V-J junction (deletion=attempted λ rearrangement), LOC96610 and PRAME (distal to VPREB1, among λ-light chain variable genes), VPREB1, and VPREB1 enhancer (proximal to VPREB1, among λ-light chain variable genes). As predicted for normal light chain rearrangement, we observed no deletions in monocytes; 100% of κ-sorted B-cells had κ V-J deletions without lambda deletions; and 100% of λ-sorted B-cells had both κ V-J and λ V-J deletions. We also analyzed 10 mature B-cell (Burkitt) lymphoma samples and observed VPREB1 deletions only in lambda-expressing samples with both κ and λ V-J deletions, and deletions always extended contiguously downstream to λ chain V-J region. We re-analyzed 30 Pre-B ALL samples (Utah cohort) and 11 Pre-B ALL cell lines by qPCR. Results validated MIP CNA patterns and differed from normal sorted B-cell and mature B-cell (Burkitt) results, displaying several types of rearrangements: 1) No light chain rearrangements with no VPREB1/enhancer deletion (n=5/30 [17%] clinical samples and 2/11 [18%] Pre-B cell lines), 2) Normal light chain (κ or κ/λ) rearrangement with no VPREB1/enhancer deletion (n=5/30 [17%] samples and 6/11 [55%] cell lines), 3) Normal light chain (κ or κ/λ) rearrangements with contiguous VPREB1/enhancer deletion (n=1/30 [3%] samples and no cell lines), 4) λ-light chain rearrangement with non-contiguous VPREB1/enhancer deletion (n=13/30 [43%] samples and 2/11 [18%] cell lines), and 5) No λ light chain rearrangement and focal VPREB1/enhancer deletion (n=6/30 [20%] samples and 1/11 [9%] cell lines). In the Utah Pre-B ALL cohort (n=47), VPREB1/enhancer deletions were significantly associated (Fisher's Exact Test, 2-tailed) with Hispanic ethnicity (p= 0.013), relapse (p= 0.027), death (p=0.026), and Day 14 M2 Marrow (5-25% blasts; p=0.038). Finally, we examined the publicly available St. Jude Children's Research Hospital ALL dataset (NEJM 351:533) and found VPREB1 under-expression associated with higher LC50 (resistance) for DNR (p=0.04) and VCR (p=0.04), but not PRED or ASP. In summary, we have shown VPREB1 deletions occur outside the context of normal light chain rearrangement. Unlike physiologic λ rearrangements, VPREB1 deletions do not always extend to the λ V-J junction in pALL. We also identified a focal, homozygous deletion just proximal to VPREB1 that may affect a possible enhancer region for this B-cell developmental gene. VPREB1/enhancer deletions were the only B-cell developmental gene lost in 25% of our cohort, perhaps defining a new Pre-B ALL subtype. Finally, deletion of VPREB1 (and its possible enhancer) predict worse clinical outcome and VPREB1 down-regulation correlates with in vitro drug resistance. Further studies of VPREB1‘s function in pediatric ALL may improve our understanding of leukemogenesis and could further refine clinical risk stratification. Disclosures:No relevant conflicts of interest to declare.

SYK Is a Tumor Suppressor In Pre-B Cell Acute Lymphoblastic Leukemia and Not a Therapeutic Target

AbstractAbstract 4199 Background:The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ-chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). We recently established that the pre-B cell receptor functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL) including ALL cells carrying the BCR-ABL1 oncogene (Trageser et al., J Exp Med, 2009). In virtually all cases of BCR-ABL1 ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. Given that the SYK tyrosine kinase represents a critical signaling molecule in the pre-B cell receptor pathway, one would expect that SYK tyrosine kinase activity has a tumor suppressive effect. It therefore seems counterintuitive that pharmacologic targeting of SYK was recently proposed as a new treatment approach for pre-B ALL (Uckun et al., Br J Haematol. 2010). While there is solid evidence for a role of Syk as a target in B cell lymphoma (Friedberg et al., Blood 2010) and B cell lineage CLL (Buchner et al., Blood 2010), where tonic B cell receptor signaling delivers critical survival signals, the role of Syk downstream of the pre-B cell receptor in ALL is unclear. Results:To clarify the role of SYK downstream of the pre-B cell receptor in pre-B ALL, we performed a genetic experiment to inducibly delete the Syk gene in pre-B ALL cells. To this end, pre-B cells from Syk-fl/fl mice were propagated in the presence of IL7 and then transformed with retroviral BCR-ABL1 or MLL-ENL oncogenes. After transformation, pre-B leukemia cells were transduced with 4-hydroxy-tamoxyfen (4-OHT)-inducible retroviral Cre-ERT2 or an ERT2 empty vector control. After puromycin-selection of Cre-ERT2 and ERT2 transduced leukemia cells, Cre-ERT2 or the ERT2 control were induced by addition of 4-OHT and deletion of Syk was studied at different time points. As assessed by Western blot and PCR, deletion of Syk was near complete after two days and undetectable after six days. We then studied changes in cell viability upon inducible deletion of Syk: Acute deletion of the Syk tyrosine kinase had no significant impact on the viability of pre-B ALL cells, even after prolonged cell culture over several weeks. We then reasoned that the effect of Syk-deletion may be subtle yet important, so we studied in BCR-ABL1-transformed Syk-fl/fl pre-B leukemia cells whether Syk-deletion sensitizes to Imatinib-treatment. Deletion of Syk was again confirmed by Western blot, yet the dose-response curves to Imatinib-treatment were superimposable for Syk-fl/fl and Syk-del/del pre-B leukemia cells. We conclude that SYK does not contribute important survival signals in our mouse model for pre-B ALL, nor does deletion of Syk sensitize BCR-ABL1-driven pre-B leukemia cells to Imatinib-treatment.We next investigated the counter-hypothesis that Syk functions as a tumor suppressor downstream of the pre-B cell receptor. To test this possibility, we tested the effect of forced pre-B cell receptor expression in the presence or absence of Syk. Syk-fl/fl and Syk-del/del pre-B leukemia cells were transduced with CD8/μ-chain or a CD8 empty vector control. The μ-chain represents the central component of the pre-B cell receptor. Forced expression of the CD8 empty vector control had no effect regardless of whether Syk was deleted or not. When pre-B cell receptor signaling was reconstituted in Syk-fl/fl cells by expression of CD/μ-chain, viability of the leukemia decreased by >80%. By contrast, deletion of Syk greatly attenuated the tumor suppressive effect of CD8/μ-chain expression and less than 25% of the leukemia cells underwent cell cycle arrest and cell death. Background:Genetic deletion of Syk unequivocally demonstrates that Syk does not deliver critical survival signals downstream of the pre-B cell receptor in ALL. This is unlike B cell lymphoma, where tonic signaling from the B cell receptor promotes cell survival via Syk (Friedberg et al., 2010; Buchner et al., 2010). On the contrary, in pre-B ALL, the Syk kinase mediates pre-B cell receptor-induced cell cycle arrest. These findings are in direct conflict with a recent report on the therapeutic usefulness of pharmacological inhibition of Syk in pre-B ALL cell lines (Uckun et al., Br J Haematol.; 149: 508-17; 2010). The compound (C-61) used in this study may have unrecognized off-target effects, which might account for the discrepancies. Disclosures:No relevant conflicts of interest to declare.

Dense Methylation of Types 1 and 2 Regulatory Regions of the CD10 Gene Promoter in Infant Acute Lymphoblastic Leukemia With MLL/AF4 Fusion Gene

Infant acute lymphoblastic leukemia (ALL) displays distinct biologic and clinical features with a poor prognosis. The CD10-negative immunophenotype of infant ALL is a hallmark and provides a predictable signature of mixed-lineage leukemia (MLL) rearrangement. Although CD10 negativity reflects an earlier stage of B-cell development, complete IgH gene rearrangements (VDJH), found in almost half of the patients, show more mature IgH status. Discordance between immunophenotype and genotype of infant ALL suggests an aberrant process in immunophenotypic steps of differentiation or a secondary down-regulation of CD10 expression. In this study, CD10-negative infant ALL with MLL/AF4, CD10-positive infant ALL with germline MLL, CD10-positive pre-B ALL cell line, infant acute myeloid leukemia (AML; M5) with MLL/AF9 and pediatric AML (M2) with AML1/ETO were analyzed for VDJH status and methylation of CD10 gene promoters. Three of the 4 infant ALL samples showed complete rearrangements of the VDJH gene with productive joints. Bisulfite sequencing of CD10 type 1 and 2 promoters showed that more than 84% of the cytosine-phosphate-guanine (CpG) dinucleotides identified were methylated in all 3 CD10-negative infant ALL samples with MLL/AF4. The CpG dinucleotides distributed in the clusters of putative Sp1-binding sites and functionally active regulatory regions of the promoters were fully methylated. In contrast, none of the CpG dinucleotides were methylated in the CD10-positive ALL samples. Structural evidence of dense methylation in the CD10 gene promoter suggested that methylated transcription factor binding sites contribute to CD10 silencing as an epigenetic mechanism.

BTG1 Controls Glucocorticoid Sensitivity in Acute Lymphoblastic Leukemia by Regulating Nuclear Receptor-Mediated Transcription.

Glucocorticoids (GCs) are global regulators of gene transcription that act by binding to the glucocorticoid receptor, a ligand-activated transcription factor belonging to the nuclear receptor family. As synthetic GCs such as prednisolone or dexamethasone specifically induce cell cycle arrest and apoptosis in lymphoid cells, these drugs are widely used in the treatment of (childhood) acute lymphoblastic leukemia (ALL). GC resistance is a strong independent predictor of poor prognosis in the treatment of childhood ALL, but the underlying mechanisms remain poorly understood. In a previous study (Kuiper et al. Leukemia 2007) we determined by array-based SNP analysis that microdeletions affecting the anti-proliferative gene B-cell translocation gene 1 (BTG1) occurred in 4 out of 33 pre-B ALL cases. In our current study we show that prednisolone causes a 3-fold induction of BTG1 mRNA expression both in pre-B ALL cell lines and primary ALL, implicating BTG1 in cellular responses to GCs. Moreover, RNA-interference-mediated knockdown of BTG1 renders RS4;11 pre-B leukemia cells refractory to prednisolone-induced apoptosis. We show by genome-wide mRNA profiling that loss of BTG1 completely abrogates prednisolone-induced gene expression by downregulation of the glucocorticoid receptor NR3C1. Moreover, we present evidence showing that BTG1 is part of a nuclear receptor complex that regulates expression of NR3C1 and consequently GC responsiveness in ALL. In conclusion, we have identified BTG1 as an important determinant of GC sensitivity in ALL.

A Therapeutic Approach for Acute Leukemias: Glycolysis Inhibition Revisited

Background: Recent evidence suggests that myeloid and lymphoid leukemia cells are dependent on aerobic glycolysis for ATP production, known as the Warburg effect. Early glycolysis inhibitors were effective but only at high mM concentrations. The novel third generation glycolysis inhibitor, 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), has shown efficacy in models of neuroblastoma, glioblastoma, colon carcinoma, and lymphoma in the mcM range and has improved oral bioavailability and biodistribution. We proposed that potent inhibition of glycolysis would lead to ATP depletion and induce growth arrest and cell death in acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) cells. Objective: To determine the feasibility and effect of 3-BrOP on human AML, precursor-B, and T cell leukemia cell lines and patient samples.Design/Methods: A panel of fifteen human pre-B ALL, T-ALL, and AML cell lines were treated with 3-BrOP to determine IC50s via cell counts, cell cycle, and Alamarblue. In addition, western blots and ATP assays were performed to determine caspase activation and ATP depletion.Results: All cell lines demonstrate growth arrest, decreased alamar blue metabolism and significant apoptosis when treated with 3-BrOP. The five human precursor-B ALL cell lines had IC50s between 10–30mcM, while the T ALL cell lines had IC50s between 15–30mcM. The human AML cell line panel had IC50s between 20–40mcM. In addition, patient samples representing ALL (IC50 15 mcM) and AML (IC50 30mcM) were sensitive to 3-BrOP mediated cell death. Of note, caspase-3 cleavage was detected by 2 hours of exposure, and was nearly complete by 6 hours in both pre-B and T-ALL suggesting a potent an early pro-apoptotic signal. Overall, cell cycle analysis at 48 hours of treatment, showed the majority of cells had died (83–97%, p<0.001). The remaining cells were apoptotic with >80% of Nalm6 (pre-B ALL) and 72% of U937 (AML) cells having sub-G0 DNA content (p<0.001). As expected, exposure to 3-BrOP reduced intracellular ATP concentrations in a dose and time dependent manner. In Nalm6 cells (pre-B ALL), by 48 hours of exposure, the ATP levels were 50% of control at a dose of 15 mcM. At a dose of 22.5 mcM, the ATP level was 13% of control, and by 30 mcM there was no detectable ATP. Importantly, at higher doses (>30mcM) ATP levels were reduced significantly within 24 hours. These effects on ATP levels correlate with dose and time dependent viability.Conclusions: These data demonstrate that a broad range of acute leukemia cell lines and patient samples appear to be dependent on glycolysis for ATP production and survival. The novel 3rd generation glycolysis inhibitor, 3-BrOP, induces cell death across all subtypes of human acute leukemia cell lines. Glycolysis inhibition via 3-BrOP represents a novel and clinically feasible therapeutic approach for acute leukemias and warrants further pre-clinical evaluation.

Dexamethasone-induced apoptosis in acute lymphoblastic leukemia involves differential regulation of Bcl-2 family members

The mechanism of glucocorticoid -induced apoptosis is not fully understood and early predictive assays based on apoptotic markers for clinical outcome in acute lymphoblastic leukemia (ALL) are scarce. The aim of this study was to characterize the involvement of Bcl-2 family members and caspase activation in dexamethasone(Dex)-induced apoptosis in ALL. Primary childhood ALL samples, the pre-B ALL cell line RS(4;11), and the T-ALL cell line CCRF-CEM were used. The involvement of Bcl-2 family members was evaluated by flow cytometry, immunocytochemistry, and western and northern blotting. Apoptosis was analyzed by annexin V and TMRE staining. Caspase activity was evaluated by a fluorometric assay. Dex induced significant down-regulation of the anti-apoptotic Bcl-2 family members Bcl-2 and Bcl-xL, differential activation of the pro-apoptotic Bak and Bax, loss of Delta psi m and cytochrome c release. Dex-induced apoptosis also involved early activation of caspases 2 and -3. Inhibition of caspase activity did not, however, protect against Dex-induced Bak/Bax activation, loss of Delta psi m or cell death. In 12 primary ALL samples Dex-induced apoptosis was associated with activation of Bax (p=0.045) and down-regulation of Bcl-2 (p=0.016) and/or Bcl-xL (p=0.004). Furthermore, ex vivo Dex-sensitivity was associated with an early treatment response to polychemotherapy (p=0.026). The differential regulation of pro- and anti-apoptotic Bcl-2 family members appears to be a key event in the execution of Dex-induced apoptosis in ALL cell lines, and also indicates a role for these proteins in primary ALL cells.