Primary pre-B Acute Lymphoblastic Leukemia Research Articles

Characterizing Deformability of Drug Resistant Patient-Derived Acute Lymphoblastic Leukemia (ALL) Cells Using Acoustic Tweezers

The role of cell mechanics in cancer cells is a novel research area that has resulted in the identification of new mechanisms of therapy resistance. Single beam acoustic (SBA) tweezers are a promising technology for the quantification of the mechanical phenotype of cells. Our previous study showed that SBA tweezers can be used to quantify the deformability of adherent breast cancer cell lines. The physical properties of patient-derived (primary) pre-B acute lymphoblastic leukemia (ALL) cells involved in chemotherapeutic resistance have not been widely investigated. Here, we demonstrate the feasibility of analyzing primary pre-B ALL cells from four cases using SBA tweezers. ALL cells showed increased deformability with increasing acoustic pressure of the SBA tweezers. Moreover, ALL cells that are resistant to chemotherapeutic drugs were more deformable than were untreated ALL cells. We demonstrated that SBA tweezers can quantify the deformability of nonadherent leukemia cells and discriminate this mechanical phenotype in chemotherapy-resistant leukemia cells in a contact- and label-free manner.

AVA4746, an Orally Available, Clinical Grade Antagonist of Integrin Alpha 4, Sensitizes Pre-B Cell Acute Lymphoblastic Leukemia to Chemotherapy

Background. Even though remarkable progress has been made in the treatment of childhood acute lymphoblastic leukemia (ALL), salvage of relapse patients remains a challenge. The role of the bone marrow (BM) microenvironment is critical to protect leukemia cells from chemotherapy. The BM microenvironment promotes cell adhesion-mediated drug resistance (CAM-DR) in ALL.We and others have shown that the adhesion molecule integrin α4, referred to hereafter as α4, mediates drug resistance of B-ALL. In our previous studies, we showed that both α4 blockade by natalizumab and inhibition by the small molecule α4 antagonist TBC3486 can sensitize relapsed ALL cells to chemotherapy. However, no α4 targeting therapy is currently clinically available to treat leukemia. Here, we preclinically evaluate a novel non-peptidic small molecule antagonist, AVA4746, which has been safely used in clinical studies, as a potential new approach to combat drug resistant ALL.Method. Six refractory or relapsed primary pre-B ALL cases were used for in vitro studies. Viability was assessed by trypan blue counts or annexin V/7AAD flow cytometric analysis and metabolic activity was evaluated by Cytoscan WST-1 assay. For in vivo evaluation a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL (LAX7R) was used.AVA4746 (15mg/kg) was administered by oral gavage twice a day continuously for 14 days, and vincristine, dexamethasone, L-asparaginase (VDL) was given intraperitoneally (weekly) for 4 weeks. Overall survival was determined by Kaplan-Meier Survival analysis.Results. AVA4746 caused a significant decrease in mean fluorescence intensity (MFI) of α4 expression in six out of six ALL cases at doses of both 5μM and 25μM after 24 hours and 96 hours compared to DMSO control. Interestingly, decreased protein expression of α4 was also observed by Western Blot analysis all six ALL cases. We tested next in two cases (LAX53, ICN13), if AVA4746 de-adheres ALL cells from its counter receptor VCAM-1. The percentages of adherence after treatment with AVA4746 (25μM) were significantly lower than after DMSO treatment (10.3%±4.9% vs. 99.9%±7.6%, p= 0.00007 for LAX7R; 8.1%±1.0% vs. 100.1%±13.6%, p= 0.0003 for LAX53; 9.0%±1.6% vs. 100.0%±14.0%, p=0.0004 for ICN13). AVA4746 was not associated with apoptosis in vitro alone or in combination with chemotherapy (VDL). Metabolic activity as assessed by WST-1 assay was markedly decreased by AVA4746 in two of two ALL cases. AVA4746 also decreased ALL proliferation in two out of two ALL samples tested. In vivo, AVA4746 in combination with VDL chemotherapy treatment led to significant prolongation of overall survival (n=6) compared with the VDL only treated group (n=6) (MST= 78.5 days vs MST= 68 days; P<0.05). There was no significant difference in survival between the PBS control group (n=5) and the AVA4746 mono-treatment group (n=5) (MST=38days vs MST= 38days).Conclusion. We have identified α4 as a central adhesion molecule in CAM-DR of ALL and have shown that AVA-4746, an orally available and specific α4 antagonist, which has been safely used in clinical studies, downregulates α4 in primary ALL and functionally de-adheres them from VCAM-1. Critically, we demonstrated that inhibition of α4 in combination with standard chemotherapy can prolong the survival of NSG mice bearing pre-B ALL. These data support further study of inhibition of α4 using AVA4746 as a novel strategy to treat drug resistant B lineage ALL. DisclosuresBhojwani:Amgen: Other: Blinatumumab global pediatric advisory board 2015. Wayne:Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Kite Pharma: Honoraria, Other: Travel support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Kim:Antisense Therapeutics Ltd: Patents & Royalties.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate.

The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia-targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MAX dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-CD22 antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2mg/kg Ab for 6 doses - twice a week for 3 weeks) more than doubled the mouse survival time in both Reh (median survival time 20.5 vs. 42.5 days, p<0.001) and primary preB ALL (median survival time 29.3 vs. 63 days, p<0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.

PI3Kdelta Inhibitor, CAL-101, De-Adheres Primary Pre-B ALL from VCAM-1 and Induces Apoptosis in Primary Pre-B ALL

BACKGROUND: Relapse remains a major problem in the treatment pre-B acute lymphoblastic leukemia (ALL). ALL cells are physically anchored in the bone marrow (BM) microenvironment through a network of adhesion molecules. Adhesion also creates intracellular signals that regulate proliferation and cell death. We have previously identified integrin α4 (α4) as a critical molecule for such cell-adhesion-mediated drug resistance (CAM-DR) in pre-B ALL. In chronic lymphocytic leukemia (CLL), α4-mediated activation of the PI3K/AKT pathway was reported. In ALL, stromal cell protection of B-lineage ALL cells has also been shown to require active Akt. However, it remains elusive how this target can be therapeutically harnessed. Therefore, we investigated if directly targeting the PI3K/AKT pathway downstream of α4 can overcome CAM-DR in pre-B ALL using a novel, FDA-approved PI3Kδ inhibitor, CAL-101METHODS: For in vitro studies, murine integrin α4 conditional knockout mouse model (α4fl/fl) oncogenically transformed with BCR-ABL1+ and patient-derived (primary) pre-B ALL cells (Philadelphia chromosome negative) co-cultured with murine calvaria-derived mesenchymal stromal (OP9) cells were used. Annexin V/7-AAD staining was used for viability determination by flow cytometry and Western Blot was used for determination of PI3Kδ and p-Akt expression. For in vivo experiments, we used a NOD/SCID IL2Rγ-/- xenograft model of primary pre-B ALL.RESULTS: Integrin α4fl/fl cells were cultured under lymphoid-skewing conditions and oncogenically transformed using BCR-ABL1 (p210). Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which α4 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen while plated on murine VCAM-1. In the absence of a4, mouse pre-B ALL cells contain markedly reduced levels of pAKT (ser473). In a parallel experiment, we observed in primary pre-B ALL cells co-cultured with OP9 cells an increase in p-AKT compared to media-only cultured ALL cells indicating activation of the PI3K/AKT pathway upon adhesion to stroma. Under starvation conditions, primary pre-B ALL cells (LAX7R) were incubated for 4 hours without serum or stroma, and subsequently treated for 48 hours with an integrin α4 blocking antibody or with control IgG4. By Western Blot, we detected decreased levels of activated p-AKT in the cells with α4 blockade, but not with control, indicating that the AKT pathway is affected by modulation of α4.To directly target the PI3K/AKT pathway, we used CAL-101, a small molecule inhibitor highly selective for the δ isoform, which was recently FDA-approved for treatment of CLL and Non-Hodgkin Lymphoma (NHL). Previously, in primary samples from CLL patients and ALL cell lines, CAL-101 blocked PI3Kδ-AKT signaling and promoted apoptosis. However, a preclinical evaluation in primary pre-B ALL patients in vitro and in vivo are missing. For these reasons, we used CAL-101 in the following experiments as a specific PI3Kδ inhibitor in primary pre-B ALL. Expression of PI3Kδ was demonstrated by Western Blot in 8 out of 8 cases of primary pre-B ALL. After incubation of primary pre-B ALL cells with different concentrations of CAL-101 (0.1-10 µM), dose-dependent downregulation of pAkt was observed. ALL cells were plated on immobilized VCAM1 (the counter-receptor for α4) or OP9 cells and treated with vehicle control (DMSO) or CAL-101 (10 µM) for 2 days. CAL-101 de-adhered ALL cells 69.5%±0.7% from VCAM1, and only 13%±0.5% from OP-9 cells, indicating an inhibition of the of the VCAM-1 and α4 interaction. ALL cells treated with CAL-101 (10 µM) for 5 days showed a marked decrease in viability as demonstrated by AnnexinV negative and 7AAD negative staining compared to DMSO-treated controls (66.2%±1.5% vs 87.9%±1.0%, p=0.0003) indicating that mono-treatment with CAL-101-decreases viability of ALL cells. Further studies addressing whether PI3Kδ inhibition is beneficial in the setting of combination chemotherapy for ALL in vitro and in vivo are ongoing.CONCLUSION: Taken together, our data demonstrate that the AKT pathway is affected by modulation of α4. PI3Kδ inhibition with CAL-101 de-adheres pre-B ALL cells from the α4-counter-receptor VCAM1, downregulates pAkt and induces apoptosis in ALL. Data derived from further studies will determine the potential of the FDA-approved PI3Kδinhibitor CAL-101 as a novel therapy for pre-B ALL. DisclosuresWayne:MedImmune: Honoraria, Research Funding, Travel Support Other; NIH: Co-inventor on investigational products with patents, Co-inventor on investigational products with patents Other.

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.

Integrin alpha4 blockade sensitizes drug resistant pre-B acute lymphoblastic leukemia to chemotherapy

AbstractBone marrow (BM) provides chemoprotection for acute lymphoblastic leukemia (ALL) cells, contributing to lack of efficacy of current therapies. Integrin alpha4 (alpha4) mediates stromal adhesion of normal and malignant B-cell precursors, and according to gene expression analyses from 207 children with minimal residual disease, is highly associated with poorest outcome. We tested whether interference with alpha4-mediated stromal adhesion might be a new ALL treatment. Two models of leukemia were used, one genetic (conditional alpha4 ablation of BCR-ABL1 [p210+] leukemia) and one pharmacological (anti-functional alpha4 antibody treatment of primary ALL). Conditional deletion of alpha4 sensitized leukemia cell to nilotinib. Adhesion of primary pre-B ALL cells was alpha4-dependent; alpha4 blockade sensitized primary ALL cells toward chemotherapy. Chemotherapy combined with Natalizumab prolonged survival of NOD/SCID recipients of primary ALL, suggesting adjuvant alpha4 inhibition as a novel strategy for pre-B ALL.

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.

Abstract B32: A novel preclinical model to identify therapies for CRLF2 B-ALL and reduce childhood cancer health disparities.

Abstract Hispanic children with Acute Lymphoblastic Leukemia (ALL) have a 39% higher death rate than white children. A major contributor to this disparity is the lack of therapies that specifically target CRLF2 B cell ALL. This high-risk leukemia occurs five times more frequently among children of Hispanic/Latino ethnicity than others and represents the most significant biological component of childhood cancer health disparities identified to date. CRLF2 B-ALL is caused by genetic alterations that result in B cells that overexpress the cytokine receptor component, CRLF2, leading to malignant transformation and high-risk leukemia with poor prognosis. CRLF2 pairs with the IL-7Rα, to form a receptor complex that is activated by the cytokine, TSLP. Activation of the receptor complex stimulates downstream JAK-STAT5 phosphorylation that has been shown to induce proliferation and survival of B-ALL cells. Activating JAK mutations are found in many cases of CRLF2 B-ALL and have led to speculation that stimulation of the receptor by TSLP is not a factor in this disease. In preliminary studies to address this question we evaluated the effect of TSLP in CRLF2 B-ALL cells with JAK defects. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary CRLF2 B-ALL cells. These data suggest that TSLP can activate JAK-STAT5 signaling to induce downstream survival and proliferation in CRLF2-B-ALL cells, including those with JAK defects. Bone marrow (BM) provides the tumor microenvironment that can harbor chemoresistant B-ALL cells responsible for relapse in B-ALL. We used RT-PCR and ELISA assays to show that TSLP is expressed in BM cells from pediatric patients and thus could provide an in vivo source of TSLP to stimulate CRLF2 B-ALL cells. Human-mouse xenograft models produced by transplanting human leukemia cells into immune deficient mice mimic the in vivo environment and are the model of choice for identifying therapies that target the mechanisms of chemoresistance that are characteristic of high-risk leukemia. However, mouse TSLP is different from most other cytokines produced in the xenograft in that it is species-specific and does not activate the human TSLP receptor complex that contains CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interactions that our data implicate as a contributing factor in CRLF2 B-ALL. To overcome this obstacle we have engineered immune deficient mice to express human TSLP (hTSLP+ mice) as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show hTSLP levels in the hTSLP+ mice that approximate the normal range in human plasma. We used this hTSLP+/- xenograft model system to study the in vivo effects of TSLP on mice transplanted with a CRLF2 B-ALL cell line harboring a JAK defect (MUTZ5) and with primary pre-B ALL cells from a Hispanic patient. Mice were euthanized at 5 weeks and BM disease was evaluated. In recipients of MUTZ5 B-ALL cells the percentage of viable leukemia cells in hTSLP+ mice was twice that observed in hTSLP- mice. Similarly, in recipients of primary B-ALL, the percentage of viable leukemia cells was higher in hTSLP+ than hTSLP- mice. These data provide evidence that the TSLP produced in this model is active and that TSLP-CRLF2 interactions contribute in vivo to CRLF2-B-ALL. This model will be particularly important for identifying therapies that can effectively target CRLF2 B-ALL and reduce cancer health disparities in Hispanic childhood B-ALL. Citation Format: Francis L. Olivia, Shannalee R. Martinez, Terrence Bennett, Ineavely Baez, Terry-Ann Milford, Christopher L. Morris, Ross O. Fisher, Xiao-Bing Zhang, Rui-Jun Su, Sinisa Dovat, Kimberly J. Payne. A novel preclinical model to identify therapies for CRLF2 B-ALL and reduce childhood cancer health disparities. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B32.

Abstract B218: CBP/catenin antagonism inhibits self-renewal of acute lymphoblastic leukemia.

Abstract Background: Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), drug resistance in ALL remains a major problem warranting new treatment strategies. Recent studies have demonstrated that survivin, a member of the inhibitor of apoptosis (IAP) family proteins, is upregulated in ALL of relapsed patients but not in drug-sensitive ALL. The expression of survivin depends on the formation of a complex between catenin (beta or gamma) and its co-activator CBP. T-cell factor (TCF)/catenin/CBP mediated transcription is involved in self-renewal of progenitor cells. However, a switch to TCF/catenin/p300 mediated transcription, which can be induced pharmacologically with ICG-001, a novel specific inhibitor of binding to the N-terminus of CBP, is described as critical to inhibit CBP-mediated self-renewal. We hypothesized that selective suppression of CBP/catenin signaling using ICG-001 offers a promising therapeutic principle to eradicate drug resistant ALL and showed previously that ICG-001 can sensitize patient-derived (primary) pre-B ALL cells to chemotherapy in vitro and in vivo. However, the underlying mechanism has not been described. Results: For this purpose, we initially determined expression of nuclear and cytosolic β- and γ-catenin expression in 13 primary ALL cases encompassing various cytogenetic aberrations by Western blot. Primary pre-B ALL cells were treated with and without ICG-001 and co-cultured with a murine stromal layer (OP9 cells) for 48 hours. Co-Immunoprecipitation showed that the binding of β- and γ-catenin with CBP, but not with p300, was inhibited by ICG-001 indicating that ICG-001 binds CBP and selectively disrupts β- and γ-catenin/CBP interaction. To determine the effect of ICG-001 on self-renewal in ALL, we performed a colony forming (CFU) assay with two primary ALL cells (LAX7R, SFO2) treated with single agent ICG-001 (10 m and 25 m). LAX7R showed a significant reduction with ICG-001 treatment (10 m and 25 m) compared with media only group (331.3× 42.8 and 57×13.5 vs 698.3× 68.4; p&amp;lt;0.05) in primary platings, and there were no colonies found in secondary platings of cells treated with ICG-001 (25 m), indicating that ICG-001 abrogates self-renewal of ALL cells. Similarly SFO2 cells showed no secondary colony counts with ICG-001 treatment (25 m) whereas control cells were replatable. These experiments were repeated three times in triplicate. Preclinical in vitro evaluation of five primary ALL cells treated continuously with ICG-001 (10μM- or, 25μM) or DMSO as vehicle control with and without chemotherapy consisting of Nilotinib for Philadelphia chromosome (Ph) positive ALL and Vincristine, Dexamethasone and L-Asparaginase for Ph negative ALL, that chemotherapy alone cannot eradicate primary ALL cells. However, in combination with ICG-001, chemotherapy led to the eradication of all five primary ALL cases, indicating that CBP/catenin antagonism can overcome drug resistance in ALL. Conclusion: Taken together, our data shows that targeting CBP/β- and γ-catenin by the small molecule inhibitor ICG-001 can abrogate primary ALL by inhibition of self-renewal of ALL cells. These findings provide the basis for a new treatment approach against drug resistant ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B218.

Adjuvant CD49d Blockade Eradicates Chemoresistant ALL

AbstractAbstract 869Despite the recent advances in chemotherapy for acute lymphoblastic leukemia (ALL), drug resistance resulting in relapse and long-term side effects of current treatments warrant new treatment modalities. Integrin α4β1 (VLA4/ITGA4/CD49d) mediates adhesion of hematopoietic cells onto bone marrow cells and has been implicated in cell adhesion-mediated drug resistance of leukemia cells. Gene expression analyses indicate that VLA4 is upregulated in B-lineage Acute Lymphocytic Leukemia (ALL). Therefore, we hypothesize that VLA4 might be a potential target for treatment of drug resistant ALL To test our hypothesis, we determined the effect of VLA4 inhibition on engraftment of primary pre-B ALLs using a humanized CD49d antibody, Tysabri, as a single agent in our NOD/SCID xenograft model of primary pre-B ALL. Tysabri is known to mobilize normal hematopoietic progenitor cells into the circulation. It blocks binding of VLA-4 to its counter receptors VCAM-1 and osteopontin and we have shown previously in a small pilot study that adjuvant administration with chemotherapy sensitizes one drug resistant primary ALL in vivo to drug treatment. In this study, we injected primary ALL cells from eight different donors into NOD/SCID mice. The samples encompass various cytogenetic aberrations (BCR-ABL, E2A-PBX, MLL-AF, normal karyotype). Cells were luciferase-transduced for in vivo cell tracking and pretreated in vitro with either Tysabri (n=3 per leukemia, n=24 total) or human Ig as a control (n=3 per leukemia, n=24 total). Recipients of Tysabri treated leukemias showed significantly prolonged median survival time (BCR-ABL: MST=112days, E2A-PBX: MST=83days, MLL-AF4: MST=51days; Normal karyotype: MST=48days) compared to control groups (BCR-ABL: MST=84days, E2A-PBX: MST=54days, MLL-AF4: MST=35days; Normal: MST=39days) (p<0.05). Therefore, engraftment of leukemia was significantly delayed in the Tysabri-treated groups as determined by bioluminescent imaging (p<0.05) and survival analysis (p<0.05). Next, we injected two luciferase-labeled pre-B ALLs (US7R, RS4;11) into NOD/SCID mice, which were then treated intraperitoneally with saline (US7R: n=4; RS4;11: n=3), Tysabri (US7R: n=4; RS4;11: n=3), VDL (Vincristine, Dexamethasone and L-Asparaginase) (US7R: n=9; RS4;11: n=5), or VDL+Tysabri (US7R: n=9; RS4;11: n=5), for 4 weeks. Tysabri-treated groups showed prolonged survival time (US7R: MST=52days; RS4;11: MST=83) compared with saline-treated groups (US7R: MST=38days; RS4;11: MST=60 days) (p=0.007). VDL-only treated animals died rapidly (US7R: MST=74days; RS4;11: MST=109 days), however, the animals treated with the combination VDL+ Tysabri, survived disease-free until the end of follow-up (US7R: MST=151days; RS4;11: MST=141 days) (p<0.0001). The sacrificed animals showed absence of human CD45 in spleen, liver, bone marrow and lung by immunohistochemistry and flow cytometry indicating eradication of recalcitrant leukemia cells. We have also shown in vivo using an immunocompetent mouse model that VLA4 ablation does not result in dose-limiting toxicity to normal hematopoietic cells after VDL or 5-FU treatment. To understand further the role of VLA4 deletion in ALL, we established a model of murine leukemia using bone marrow cells from VLA4 floxed mice, retrovirally transformed with BCR-ABL1 p210 and cmyc. Subsequent to leukemic outgrowth, cells were transduced with either Empty GFP control, or Cre-GFP vector to delete VLA4. Knockout of VLA4 in transduced cells was detected by PCR on genomic DNA and by flow cytometry (Empty GFP control: 97% CD49+; Cre-GFP vector: 0.8% CD49+). Upon in vitro culturing of the cells 4-fold more VLA4 deleted cells were found in the supernatant compared to the control cells (p<0.05) determined by Trypan blue exclusion counts of dead cells, indicating that CD49d in murine leukemia is required for cell adhesion. Further functional studies addressing engraftment and gene expression upon induced VLA4 deletion are ongoing. Taken together, our data demonstrate that CD49d-blockade with adjuvant chemotherapy can eradicate chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant inhibition of integrins to overcome relapse of leukemia. Disclosures:No relevant conflicts of interest to declare.

Evaluation of CD9 as a Candidate Leukemia Stem Cell Marker In Childhood Precursor B Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3241Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures:No relevant conflicts of interest to declare.

Histone Profiling of Normal B-Precursors and Primary Pre-B Acute Lymphoblastic Leukemia Reveals Distinct Aberrant Histone Codes In MLL-Rearranged Vs. Wild Type MLL Leukemias That Correlate with Differential Expression of Key MLL Target Genes

Abstract 2503 Epigenetic regulation of gene transcription is mediated both by methylation of DNA CpG islands and the local configuration of chromatin, which is dynamically regulated by post-translational modifications, or “marks”, of key lysines (K) of histones (especially H3). Some marks are associated with transcriptional repression [trimethylation (me3) of K9 and K27], and some with activation [me3 of K4, dimethylation (me2) of K79 and acetylation (Ac) of K9 and K14]. The MLL gene encodes a protein that functions as a master regulator of target gene expression by methylating H3K4 via its SET domain, and by interacting with other proteins with histone modifying properties. MLL is frequently rearranged (MLL-r) by translocations in acute leukemias, which exhibit a distinct global gene expression pattern. Many MLL-r partner genes form complexes that can methylate H3K79. Thus, histone modifications may be central to the function of both wild type (MLL-wt) and MLL-r. We hypothesized that aberrant histone coding of target genes contributes to MLL-r leukemogenesis. We characterized the histone code associated with the promoters of selected genes in n=5 MLL-r pre-B ALL samples (MLL-AF4 or MLL-ENL), n=4 MLL-wt pre-B ALL samples (TEL-AML1 or hyperdiploid) and normal control B-precursors (CD19+ cord blood cells). We selected 9 genes differentially overexpressed in MLL-r leukemia (HOXA7, HOXA9, MEIS1, FLT3, CCNA1, ZC3H12C, ATP8B4, C20orf103, and PROM1), and 3 control genes that are not MLL targets (HOXA1, HOXC8, LTF). We performed ChIP with antibodies specific for key H3 modifications (K4me3, K9me3, K9/14Ac, K27me3 and K79me2), followed by qPCR for the selected genes. Expression was measured by RT/qPCR. All 9 MLL target genes were significantly overexpressed in the MLL-r cohort, and this was associated with a specific “activating” histone code at the genes' promoters (fig 1 – MEIS1, e.g.). The opposite “repressive” code was found in the MLL-wt cohort, and in the MLL-r cohort at the promoters of the control genes. Compared to both sets of leukemias, normal B-precursors exhibited a paucity of histone modifications for all genes. For most genes, a specific developmental pattern of alterations in the histone code and corresponding relative change in expression could be traced from the normal B-precursors to the leukemia cells. This pattern was strikingly different in MLL-r leukemias than in MLL-wt leukemias, suggesting that the acquisition of MLL-r by normal B-precursors causes altered gene expression patterns via changes in the histone code. For most genes, normal B-precursors exhibit both the activating K4me3 mark and the repressive K27me3 mark, and express low but detectable levels of RNA. In MLL-r leukemias, upregulation of genes is associated with an increase in K4me3, loss of K27me3, and gain of K9/14Ac and/or K79me2. In MLL-wt leukemias, silencing of genes is associated with loss of K4me3 and gain of K9me3. To study the direct effects of MLL-wt and MLL-r on the histone code, we used 2 rounds of siRNA over 48 hours to knock down MLL-AF4 only, MLL-wt only or both in the RS4;11 cell line (MLL-AF4+ B-precursor ALL), then performed RT/qPCR and ChIP/qPCR. We achieved at least 60% knock down of MLL-AF4 and/or MLL-wt. Knock down of MLL-wt, with or without concomitant knock down of MLL-AF4, did not diminish the K4me3 mark for any genes, suggesting that MLL's SET domain is not required to maintain K4 methylation. While knock down of MLL-AF4 or MLL-wt alone did not diminish K79 methylation, knock down of both completely removed the K79me2 mark from all genes, suggesting that expression of either MLL-wt or MLL-AF4 is absolutely required for H3K79 methyltransferase activity. Two genes (HOXA7 and PROM1) demonstrated evidence of direct transcriptional regulation by MLL-AF4, since their expression decreased markedly after knock down of MLL-AF4 alone or with MLL-wt, but not with MLL-wt alone. In summary, primary MLL-r pre-B ALLs exhibit a distinct activating histone code at key overexpressed target genes when compared to MLL-wt pre-B ALLs and normal B-precursors. A causative role for MLL fusion proteins is suggested by the distinct pattern of histone code progression from normal B-precursors to MLL-r leukemias. Furthermore, knock down experiments provide direct evidence that some of the observed histone modifications in MLL-r leukemia, particularly H3K79 methylation, are directly downstream of wild type and mutant MLL. Disclosures: No relevant conflicts of interest to declare.