Human pre-B Acute Lymphoblastic Leukemia Research Articles

Targeting TNF/IL-17/MAPK pathway in hE2A-PBX1 leukemia: effects of OUL35, KJ-Pyr-9, and CID44216842.

t(1;19)(q23;p13) is one of the most common translocation genes in childhood acute lymphoblastic leukemia (ALL) and is also present in acute myeloid leukemia (AML) and mixed-phenotype acute leukemia (MPAL). This translocation results in the formation of the oncogenic E2A-PBX1 fusion protein, which contains a trans-activating domain from E2A and a DNA-binding homologous domain from PBX1. Despite its clear oncogenic potential, the pathogenesis of E2A-PBX1 fusion protein is not fully understood (especially in leukemias other than ALL), and effective targeted clinical therapies have not been developed. To address this, we established a stable and heritable zebrafish line expressing human E2A-PBX1 (hE2APBX1) for high-throughput drug screening. Blood phenotype analysis showed that hE2APBX1 expression induced myeloid hyperplasia by increasing myeloid differentiation propensity of hematopoietic stem cells (HSPCs) and myeloid proliferation in larvae, and progressed to AML in adults. Mechanistic studies revealed that hE2A-PBX1 activated the TNF/IL-17/MAPK signaling pathway in blood cells and induced myeloid hyperplasia by upregulating the expression of the runx1. Interestingly, through high-throughput drug screening, three small molecules targeting the TNF/IL-17/MAPK signaling pathway were identified, including OUL35, KJ-Pyr-9, and CID44216842, which not only alleviated the hE2A-PBX1- induced myeloid hyperplasia in zebrafish but also inhibited the growth and oncogenicity of human pre-B ALL cells with E2A-PBX1. Overall, this study provides a novel hE2A-PBX1 transgenic zebrafish leukemia model and identifies potential targeted therapeutic drugs, which may offer new insights into the treatment of E2A-PBX1 leukemia.

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

CREB knockdown inhibits growth and induces apoptosis in human pre-B acute lymphoblastic leukemia cells through inhibition of prosurvival signals.

A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation. The gene expression levels of prosurvival signals Bcl-2, Mcl-1, Bcl-xL, survivin and XIAP were down-regulated upon CREB suppression. These findings indicate a critical role for CREB in proliferation, survival, and apoptosis of BCP-ALL cells. The data also suggest that CREB could possibly serve as potential therapeutic target in BCP-ALL.

PTEN opposes negative selection and enables oncogenic transformation of pre-B cells.

PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor in many types of cancer. To test a tumor suppressive role of PTEN in pre-B acute lymphoblastic leukemia (ALL), we induced Cre-mediated deletion of Pten in mouse models of pre-B ALL. In contrast to its role as a tumor suppressor in other cancers, loss of one or both alleles of Pten caused rapid cell death of pre-B ALL cells and was sufficient to clear transplant recipient mice of leukemia. Small molecule inhibition of PTEN in human pre-B ALL cells resulted in AKT hyperactivation, p53 checkpoint activation and cell death. Loss of PTEN function in pre-B ALL cells was functionally equivalent to acute activation of autoreactive pre-BCR signaling, which engaged a deletional checkpoint for removal of autoreactive B cells. We propose that targeted inhibition of PTEN and hyperactivation of AKT triggers a checkpoint for elimination of autoreactive B cells and represents a new strategy to overcome drug-resistance in human ALL.

PTEN Is Essential for Normal Cytokine Signaling and Oncogenic Transformation of Pre-B Cells

Background: PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor throughout all main types of human cancer. Recent work demonstrated that loss of PTEN in T cell acute lymphoblastic leukemia (ALL) confers glucocorticoid resistance. To develop a model for glucocorticoid resistance in pre-B ALL, we studied Cre-mediated deletion of Pten.Results: Surprisingly, loss of one or both alleles of Pten caused rapid cell death in pre-B ALL cells and was sufficient to cure transplant recipient mice from leukemia. Furthermore, deletion of Pten in pre-B cells disabled oncogenic transformation by BCR-ABL1 and NRASG12D oncogenes. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency across all main subtypes of mature B cell lymphoma, myeloid leukemias and T cell lineage leukemia/lymphoma (11.3% in 1,697 samples). Strikingly, however, lesions of PTEN were not detected in any of 670 pre-B ALL patient samples. Whereas high expression levels of PTEN predict favorable outcomes for patients with T-ALL, pre-B ALL patients with high PTEN expression levels at the time of diagnosis have particularly poor clinical outcomes (P=0.01). Studying the unexpected role of PTEN in B cell lineage ALL, we developed a genetic model for Cre-mediated deletion of Pten in mouse pre-B ALL cells carrying BCR-ABL1 or NRASG12D oncogenes. Deletion of one or both alleles of Pten reduced the colony forming ability (p < 10E-06) and proliferation rate (p < 10E-04) of pre-B ALL cells while strongly increasing cellular senescence (P < 10E-03). Since immediate effects of Pten deletion include hyperactivation of PI3K-AKT signaling, we hypothesized that small molecule inhibitors that limit PI3K-AKT pathway activity may rescue the pre-B ALL cells from cell death when Pten is inducibly deleted. Therefore, we tested the effect of AKT and PI3K inhibitors AZD5363 and BKM120 on Pten deleted cells and confirmed that chemical inhibition of PI3K-AKT pathway indeed rescued cell death of pre-B ALL cells upon Pten deletion. Also, global tyrosine phosphoprofiling by quantitative mass spectrometry revealed that Cre-mediated deletion of Pten strongly induced activation of CBL, a ubiquitin ligase that induces degradation of the IL7 receptor in pre-B cells and oncogenic tyrosine kinases in leukemia. Genetic experiments confirmed that deletion of Pten and hyperactivation of AKT engaged a novel, CBL-dependent pathway of negative feedback inhibition of STAT5 and tyrosine kinase signaling. Consistent with these finding, Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of pre-B ALL revealed a strong negative correlation between STAT5 protein levels and phospho-ATKT308 (P=0.0021) and phospho-AKTS473 (P=0.0005).Unlike pre-B ALL, deletion of Pten in CML like cells showed no toxicity. To further study the lineage specifity of Pten dependency we used a doxycycline-inducible CEBPa construct to differentiate pre-B cell ALL into myeloid lineage leukemia cells. Similar to our finding in CML like cells, unlike the pre-B ALL, myeloid lineage Leukemia showed no dependency on Pten and the deletion of Ptencaused no significant change in the cells viability. Studying the basal level of PTEN, pre-B cells showed ~40-fold higher expression levels than myeloid lineage cells. Consistently, genetic knockdown of PTEN using two different shRNA was only toxic in patient-derived pre-B but not myeloid leukemia cells.Clinical relevance: To find out the clinical potential of our finding, we tested the effects of SF1670 a small molecule inhibitor of PTEN on a panel of pre-B ALL and CML. Interestingly, the pre-B ALL showed more sensitivity to PTEN inhibition compared to CML cells.Conclusion: Our analysis uncovered an unexpected vulnerability of human Pre-B ALL cells toward PTEN inhibition and hyperactivation of the PI3K-AKT pathway, which provides an explanation why activating mutations of this pathway are negatively selected in human pre-B ALL. Since short (few hours) and profound inhibition of Pten is sufficient to commit human pre-B ALL cells to cell death, transient inhibition of this pathway or transient hyperactivation of PI3K-AKT may be a promising strategy to target multi-drug resistant human ALL. DisclosuresNo relevant conflicts of interest to declare.

Erk and Stat5 Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

▪Background and Hypothesis: Targeted therapy of cancer typically focuses on the development of agents that will inactivate a transforming oncogene. In this study, we tested the concept that besides the oncogene itself, factors that enable permissiveness of a normal cell to oncogenic signaling represent a novel class of therapeutic targets. This hypothesis was based on three findings. First, acute activation of oncogenes in normal pre-B cells typically caused immediate cell death, unless pre-B cells were capable of adapting quickly enough to a high level of signaling output. Second, few surviving pre-B cell clones achieved permissiveness to oncogenic signaling by strong activation of negative feedback control of Erk and Stat5. Third, robust feedback control of Erk and Stat5 distinguishes normal pre-B cells from fully transformed pre-B acute lymphoblastic leukemia (ALL) cells.Results: To test the significance of strong feedback control of Erk and Stat5 signaling in pre-B ALL cells, we developed genetic loss-of function models for six central molecules in Erk (DUSP6, SPRY2, ETV5) and Stat5 (Cish, SOCS2, SOCS3) feedback control. Genetic deletion of the sprouty family Ras inhibitor Spry2, the Erk dual specificity phosphatase Dusp6 and their transcriptional activator Etv5, decreased robustness of Erk feedback control and compromised oncogenic transformation in mouse models for pre-B ALL. Likewise, ablation of Stat5 feedback control through deletion of the suppressors of cytokine signaling (SOCS) family molecules Cish, Socs2 and Socs3 reversed permissiveness of pre-B cells. Studying deletion of Spry2 (Erk) and Cish (Stat5) in an in vivo transplant model using inducible, Cre-mediated deletion of Spry2 and Cish in pre-B ALL cells confirmed that Erk and Stat5 feedback control are essential for malignant transformation and development of lethal leukemia. Genetic deletion of Erk (Dusp6, Spry2, Etv5) and Stat5 (Cish, Socs2, Socs3) feedback control impairs leukemic transformation of pre-B cells.Searching for factors that restrict permissiveness to oncogene signaling, we identified the pre-B cell tumor suppressor IKZF1, which is deleted in a large fraction of pre-B ALL cases. IKZF1 directly bound to and transcriptionally repressed multiple promoters of Erk and Stat5 feedback control and IKZF1 deletion raised the limit of maximum allowable oncogene signaling strength in pre-B ALL cells. We propose that the pre-B cell tumor suppressor IKZF1 functions as transcriptional repressor of Erk and Stat5 feedback control and thereby retains pre-B cells in a Non-permissive state.Clinical relevance: To assess potential usefulness of this finding for the development of future treatment strategies, we tested the effect of a specific small molecule inhibitor of DUSP6, E-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), which was designed as an allosteric inhibitor of the interaction between DUSP6 and phospho-ERK1/2. Interestingly, BCI acutely subverted Erk feedback control and selectively induced cell death in pre-B ALL cells. Small molecule inhibition of DUSP6 was sufficient to overcome conventional mechanisms of drug-resistance in pre-B ALL and selectively killed patient-derived pre-B ALL cells in a leukemia transplant model. BCI treatment, similar to Dusp6-deletion in our leukemia mouse model, led to the accumulation of P53 and ARF in patient-derived pre-B ALL cells. In addition, small molecule inhibition of DUSP6 had strong selective activity on drug-resistant patient-derived pre-B ALL cells that were injected into NOD/SCID transplant recipient mice. These findings identify permissive negative feedback control of oncogenic signaling as a previously unrecognized vulnerability of pre-B ALL cells and a new class of potential therapeutic targets.Conclusion: Targeting negative feedback regulation of both Erk and Stat5 signaling for the treatment of pre-B ALL seems counter-intuitive because it represents effectively the opposite of current efforts of targeted inhibition of oncogenic signaling. Our results, however, demonstrate that a robust negative feedback regulation is required for the leukemic transformation and development of fatal leukemia in pre-B ALL. We demonstrate that feedback control of Erk and Stat5 signaling represents a previously unrecognized vulnerability and, potentially, a novel class of therapeutic targets in human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Mouse xenograft modeling of human adult acute lymphoblastic leukemia provides mechanistic insights into adult LIC biology

AbstractThe distinct nature of acute lymphoblastic leukemia (ALL) in adults, evidenced by inferior treatment outcome and different genetic landscape, mandates specific studies of disease-initiating mechanisms. In this study, we used NOD/LtSz-scid IL2Rγ nullc (NSG) mouse xenotransplantation approaches to elucidate leukemia-initiating cell (LIC) biology in primary adult precursor B (pre-B) ALL to optimize disease modeling. In contrast with xenografting studies of pediatric ALL, we found that modification of the NSG host environment using preconditioning total body irradiation (TBI) was indispensable for efficient engraftment of adult non-t(4;11) pre-B ALL, whereas t(4;11) pre-B ALL was successfully reconstituted without this adaptation. Furthermore, TBI-based xenotransplantation of non-t(4;11) pre-B ALL enabled detection of a high frequency of LICs (<1:6900) and permitted frank leukemic engraftment from a remission sample containing drug-resistant minimal residual disease. Investigation of TBI-sensitive stromal-derived factor-1/chemokine receptor type 4 signaling revealed greater functional dependence of non-t(4;11) pre-B ALL on this niche-based interaction, providing a possible basis for the differential engraftment behavior. Thus, our studies establish the optimal conditions for experimental modeling of human adult pre-B ALL and demonstrate the critical protumorogenic role of microenvironment-derived SDF-1 in regulating adult pre-B LIC activity that may present a therapeutic opportunity.

In vivo evidence that phenylalanine 171 acts as a molecular brake for translesion DNA synthesis across benzo[a]pyrene DNA adducts by human DNA polymerase κ

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/-), F171A (POLK F171A/-) or lack expression of Pol κ (POLK-/-) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (-)- or (+)-trans-anti-BPDE-N(2)-dG in the supF gene. The frequencies of mutations were in the order of POLK-/->POLK+/->POLK F171A/- both in (-)- and (+)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N(2)-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

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.

Galectin-3 in pre-B acute lymphoblastic leukemia

Galectin-3 in pre-B acute lymphoblastic leukemia

Restoration of Mismatch Repair Functions in Human Cell Line Nalm-6, Which Has High Efficiency for Gene Targeting

Gene targeting is a powerful approach in reverse genetics. The approach has been hampered in most of human cell lines, however, by the poor targeting efficiency. Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, exhibits exceptionally high gene targeting efficiency and is used in DNA repair and the related research fields. Nonetheless, usage of the cell line is still limited partly because it lacks expression of MSH2, a component of mismatch repair complex, which leads to increased genome instability. Here, we report successful restoration of MSH2 expression in Nalm-6 cells and demonstrate that the recovery does not affect the high targeting efficiency. We recovered the expression by introduction of cDNA sequences corresponding to exons 9 to 16 at downstream of exon 8 of the MSH2 gene. Endogenous exons 9 to 16 were deleted in the cell line. The MSH2 expression substantially reduced spontaneous HPRT mutation frequency. Moreover, gene targeting efficiency in the MSH2-expressing cells was similar to that in the MSH2-lacking cells. In fact, we generated heterozygously REV3L knockout and the catalytically dead mutants in the MSH2-proficient Nalm-6 cells with efficiency of 20–30%. The established cell line, Nalm-6-MSH+, is useful for reverse genetics in human cells.

O-acetylated N-acetylneuraminic acid as a novel target for therapy in human pre-B acute lymphoblastic leukemia

The development of resistance to chemotherapy is a major cause of relapse in acute lymphoblastic leukemia (ALL). Though several mechanisms associated with drug resistance have been studied in detail, the role of carbohydrate modification remains unexplored. Here, we investigated the contribution of 9-O-acetylated N-acetylneuraminic acid (Neu5Ac) to survival and drug resistance development in ALL cells. A strong induction of 9-O-acetylated Neu5Ac including 9-O-acetyl GD3 was detected in ALL cells that developed resistance against vincristine or nilotinib, drugs with distinct cytotoxic mechanisms. Removal of 9-O-acetyl residues from Neu5Ac on the cell surface by an O-acetylesterase made ALL cells more vulnerable to such drugs. Moreover, removal of intracellular and cell surface-resident 9-O-acetyl Neu5Ac by lentiviral transduction of the esterase was lethal to ALL cells in vitro even in the presence of stromal protection. Interestingly, expression of the esterase in normal fibroblasts or endothelial cells had no effect on their survival. Transplanted mice induced for expression of the O-acetylesterase in the ALL cells exhibited a reduction of leukemia to minimal cell numbers and significantly increased survival. This demonstrates that Neu5Ac 9-O-acetylation is essential for survival of these cells and suggests that Neu5Ac de-O-acetylation could be used as therapy to eradicate drug-resistant ALL cells.

P03.08 - Inhibition of Casein Kinase II (CK2) Enhances Ikaros Tumor Suppressor Activity and Shows Therapeutic Efficacy in a Preclinical Leukemia Model

Casein kinase II (CK2) promotes cell cycle progression and cellular proliferation in leukemia. Overexpression of CK2 results in the development of high-risk leukemia, that is resistant to chemotherapy. The mechanism by which CK2 promotes cellular proliferation and malignant transformation is unknown. Our published data showed that CK2 phosphorylates the Ikaros tumor suppressor protein, which results in loss of its function as a transcriptional repressor. To determine the therapeutic efficacy of CK2 inhibitors, we tested their effect on Ikaros tumor suppressor function in vitro in human pre-B acute lymphoblastic leukemia (ALL) and in vivo using a preclinical human-mouse pre-B ALL xenograft model.

Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib)

BackgroundTreatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL.ResultsWe compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival.ConclusionsPHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.

Vitamin D protects acute lymphoblastic leukemia cells from dexamethasone

Vitamin D deficiency has been linked with increased cancer risk, and vitamin D has been shown to be cytotoxic to some cancer cells in vitro. In the present study we evaluated whether vitamin D would have antiproliferative or cytotoxic effects on human pre-B acute lymphoblastic leukemia cells. Contrary to our hypotheses, calcitriol, the active form of vitamin D, had no effect on leukemia cell proliferation. Calcitriol actually had a modest effect to impair dexamethasone cytotoxicity and induction of apoptosis. Further studies are needed to evaluate the effects of vitamin D on leukemia cells in vivo.

Chronic stress enhances progression of acute lymphoblastic leukemia via β-adrenergic signaling

Clinical studies suggest that stress-related biobehavioral factors can accelerate the progression of hematopoietic cancers such as acute lymphoblastic leukemia (ALL), but it is unclear whether such effects are causal or what biological pathways mediate such effects. Given the network of sympathetic nervous system (SNS) fibers that innervates the bone marrow to regulate normal (non-leukemic) hematopoietic progenitor cells, we tested the possibility that stress-induced SNS signaling might also affect ALL progression. In an orthotopic mouse model, Nalm-6 human pre-B ALL cells were transduced with the luciferase gene for longitudinal bioluminescent imaging and injected i.v. into male SCID mice for bone marrow engraftment. Two weeks of daily restraint stress significantly enhanced ALL tumor burden and dissemination in comparison to controls, and this effect was blocked by the β-adrenergic antagonist, propranolol. Although Nalm-6 ALL cells expressed mRNA for β1- and β3-adrenergic receptors, they showed no evidence of cAMP signaling in response to norepinephrine, and norepinephrine failed to enhance Nalm-6 proliferation in vitro. These results show that chronic stress can accelerate the progression of human pre-B ALL tumor load via a β-adrenergic signaling pathway that likely involves indirect regulation of ALL biology via alterations in the function of other host cell types such as immune cells or the bone marrow microenvironment.

Synergy of Sequential and Concurrent Administration of a Deglycosylated Ricin A Chain-Containing Combined Anti-CD19 and Anti-CD22 Immunotoxin (Combotox) and Cytarabine in a Murine Model of Advanced Acute Lymphoblastic Leukemia

Abstract 1526 BACKGROUND:The immunotoxin combotox (1: 1 mixture of deglycosylated ricin-A chain (dgRTA)-containing anti-human CD19 and anti-CD22 monoclonal antibodies (MAbs) (Combotox) has demonstrated single agent activity in children (Herrera et al, 2009) and adults (Schindler et al, 2011) with relapsed/refractory B-lineage acute lymphoblastic leukemia (ALL). Even though single agent Combotox is effective in reducing the number of blasts, relapses usually occur early on especially in advanced disease. We designed experiments to explore potential synergy of concurrent and sequential administration of Combotox and cytarabine (Ara-C) in a murine model of advanced human pre-B cell ALL. MATERIALS AND METHODS: A xenograft model of advanced ALL in NOD mice was established using the human pre-B lymphoblast cell line NAML/6 (Herrera et al 2009). 6–8 week old NOD mice (Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were injected in the tail vein with 8–10×106 NALM/6 cells. The presence of leukemic blasts in the blood was confirmed microscopically 7, 14 and 18 days after inoculation. Drug administration was done after establishment of increased blast counts in order to mimic human advanced disease. In the first experiment (concurrent schedule) mice were treated daily on Days (d) 12–17 with either 1) Ara-C, 2) Combotox, 3) Ara-C concurrent with Combotox, or 4) normal saline (control), respectively. In a second experiment (sequential schedule), mice were treated with either 1) Ara-C on Days (d) 7–9 after inoculation, 2) Combotox on d14, d16 and d18, 3) Ara-C d7-9 followed by Combotox on d14, d16 and d18 or 4) normal saline (control) on d7-9, d14, d16 and d18. The doses were 200mg/kg daily x3 for Ara-C and 2.4mg/kg anti-CD19-dgRTA plus 1.5mg/kg anti-CD22-dgRTA per injection every other day x3 for Combotox The median survival time (MST) for each treatment group was calculated. Overall survival was plotted as Kaplan-Meier curve and a 2-sided log-rank test was performed. A p-value <0.05 indicated statistical significance. RESULTS: Both concurrent (Experiment #1) and sequential administration (Experiment #2) of Ara-C combined with Combotox resulted in statistically significantly improved overall survival (p-value <0.05 for both schedules). The median survival time (MST) was 27 and 28 days respectively for concurrent and sequential combined chemo-immunotherapy with Ara-C and Combotox as compared to control (MST 17 & 19 days) and/or each agent by itself (MST Ara-C 19 & 20 days; Combotox 20 and 18 days) as demonstrated in Figures 1 and 2. CONCLUSION: The combination of Ara-C and Combotox resulted in improved survival in mice xenografted with NALM/6 cells, which served as a model for advanced B-lineage ALL. Whether concurrent or sequential administration has superior efficacy is under current active investigation. These results influenced the design of a clinical trial that will explore the safety and efficacy of Ara-C combined with Combotox in adults with relapsed/refractory ALL (clinicaltrials.gov identifier NCT01408160). [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

Connective Tissue Growth Factor (CTGF) Is Essential for Self Renewal and Proliferation of Mesenchymal Stromal Cells (MSCs) and Affects Leukemia-Stromal Interactions.

AbstractAbstract 3845Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors that are critical regulators of vertebrate development. CTGF is a secreted protein that promotes extracellular matrix production, chemotaxis, cell proliferation and integrin expression. Since CTGF is highly expressed in acute lymphoblastic leukemia (ALL) and CTGF expression levels are related to ALL patient survival (Olga ST et al., Blood, 2007), we hypothesized that CTGF plays a role in regulating stromal cell proliferation and leukemia-stroma interaction. Our first goal was to characterize multipotent mesenchymal stromal cells (MSCs) from CTGF-/- mice, which die soon after birth from respiratory failure due to abnormal skeletal growth. We first attempted to isolate MSCs from BALB/C wild type and CTGF-/- newborn mice (Lvkovic S et al., Development, 2003). While we had no difficulty isolating MSCs from wild type mice, we failed to generate MSCs from CTGF-/- newborns (bone marrow, liver, spleen and thymus). As an alternative approach, we suppressed CTGF in bone marrow derived human MSCs with lenti-virus delivered CTGF shRNA (CTGF-KD-MSCs) and achieved knockdown of ≂f65% compared to vector control cells as determined by RT-PCR. Shortly after transduction (4 days) we started to observe major changes in the phenotype of CTGF-KD-MSCs, which grew 6–7 fold slower compared to vector control MSCs. CTGF-KD-MSCs displayed a significant decrease in the number of cells in S phase (from 14.7% ± 0.8% to 3.5%± 0.4%) and a concomitant increase in the number of G0/1 cells (from 68.8%±1.8% to 82.4%± 1.3%) suggesting a G0 or G1 block. To investigate if CTGF affects expression of MSC surface proteins, we analyzed standard MSC markers including CD105, CD90, CD73, CD44, CD140b, CD166 and CD45 (as a negative marker). Surprisingly, no differences in the expression of these markers in CTGF-KD-MSCs compared to vector control MSCs were observed. To determine if CTGF might regulate gene expression in MSCs, we performed a microarray analysis using Illumina arrays. By gene set analysis methods, we observed significant down regulation (p < 10-9 by the hypergeometric distribution test) in CTGF-KD-MSCs of genes involved in cell cycle progression, most notably in the Gene Ontology lists for ribosomal biogenesis and mitosis. As MSCs are known to differentiate into mesodermal cell lineages, we next tested the differentiation potential of CTGF-KD-MSCs as compared to the vector control MSCs. Following standard differentiation protocols, both CTGF-KD- and vector control-MSCs differentiated into osteoblasts and chondrocytes with no differences in their ability to differentiate into these lineages; whereas, CTGF-KD-MSCs showed 6–7 fold increase in their adipocyte differentiation potential compared to vector control MSCs. Another property of MSCs is to self-renew and generate colony forming units-fibroblast like (CFU-F). Fifty or 100 cells of either CTGF-KD-MSCs or vector control MSCs were seeded in alpha-MEM with 10% serum. After 2 weeks of culture in alpha-MEM with 10% serum, the resulting fibroblast-like colonies were counted. Vector control MSCs generated ≂f30-fold higher CFU-F compared to CTGF-KD-MSCs indicating that CTGF-KD-MSCs lack the ability to self renew. Next we determined if suppression of CTGF might impede leukemia cell migration to MSCs. Using standard trans-well assays, we examined human pre-B acute lymphoblastic leukemia (ALL) derived REH cells and found that they migrated 45% ± 4% less to CTGF-KD-MSCs compared to control MSCs. These findings suggest that CTGF plays a major role in stromal cell proliferation, self renewal and adipocyte differentiation of MSCs. In addition, CTGF is involved in leukemia cell migration towards MSCs and inhibition of CTGF impairs the migration of leukemic cells towards stromal cells and thereby provides opportunities to prevent homing of leukemic cells and sensitize them to chemotherapy. Disclosures:Andreeff:MyeloRx LLC: Consultancy, participant in research under an NCI SBIR Contract to MyeloRx LLC.

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.