Interrogating Novel Bromodomain Inhibition Resistance Mechanism in Mllr Leukemia

Abstract MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood. We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression. In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

Download Full-text

The Small Molecule Inhibitor of VLA4 TBC3486 Sensitizes Resistant ALL to Chemotherapy

Blood ◽

10.1182/blood.v120.21.1500.1500 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1500-1500 ◽

Cited By ~ 2

Author(s):

Yao-Te Hsieh ◽

Eun Ji Gang ◽

Halvard Bonig ◽

Ronald J Biediger ◽

Peter Vanderslice ◽

...

Keyword(s):

Cell Viability ◽

Small Molecule ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Chemotherapy Resistance ◽

Leukemia Cell ◽

The Novel ◽

Small Molecule Inhibition

Abstract Abstract 1500 Significant progress notwithstanding, drug resistant acute lymphoblastic leukemia (ALL) remains a therapeutic challenge, as well as acute and long-term off-target toxicity of anti-ALL therapies can be dose-limiting or debilitating. Therefore, the development of more targeted therapies is desirable. We recently provided evidence that chemotherapy resistance of ALL cells can be partly overcome by interfering with the function of VLA4, the alpha4beta1 integrin, in vivo. In those studies, we used the anti-functional antibody Natalizumab. We extended our studies to an alternative VLA4 inhibitor, the novel non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicate that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of VLA-4. As such, the compound has been shown to be efficacious in VLA-4 dependent models of inflammatory and autoimmune disease. The potential usefulness of this novel inhibitor in leukemia treatment was tested in our established in vitro and in vivo assays. LAX7R cells, primary pre-B-ALL with a normal karyotype from a patient with an early relapse, were used throughout for the studies reported here. LAX7R cells were treated with 25μM TBC3486 or THI0012 control, the inactive enantiomer of TBC3486, and seeded onto plates coated with human VCAM-1. Adhesion, scored after 2 days, was significantly inhibited by TBC3486 compared to control treated cells (7.9%±4.0 vs 95.4%±8.0; p=0.003). Proliferation rate and cell viability were unaffected by the treatments. In a co-culture system of LAX7R cells with OP9 stroma cells, which we use as an in vitro model of stroma-mediated chemotherapy resistance, we assessed differential effects of VDL (Vincristine, Dexamethasone, L-Asparaginase) on leukemia cell survival in the presence or absence of TBC3486. Stromal adhesion significantly protected LAX7R cells against VDL chemotherapy; this effect was significantly attenuated by TBC3486 compared to the control as determined by Trypan blue exclusion of dead cells (Cell viability of 39.9%±5.1 vs. 57.2±1.8; p=0.02). After these encouraging observations, we next evaluated the benefit of TBC3486 on leukemia progression in a xenotransplant assay. LAX7R cells were lentivirally labelled with luciferase for in vivo tracking and injected into NOD/SCID hosts. Three days after leukemia cell transfer, mice received either TBC3486 or THI0012 (control) (10mg/kg/d) daily for 2 weeks (intraperitoneally), with or without VDL chemotherapy. This experiment is in progress, but already survival of leukemia-bearing mice was significantly prolonged, from a median survival time (MST) for control mice of 33 days post-leukemia injection to a MST of 47 days post-leukemia injection for TBC3486 treated mice (p=0.02). Similarly, bioluminescence imaging revealed a marked delay of leukemia cell dissemination (p<0.0001). Taken together, our data demonstrate that small molecule inhibition of VLA4 using the novel TBC3486 is a suitable approach for targeting of chemotherapy-resistant leukemia. Further studies are warranted to understand and evaluate preclinically adjuvant small molecule inhibition of integrins to overcome relapse of ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Comparative Studies of B Cell Precursor ALL Pathogenesis and Translational Biology Using a Conditional E2a-PBX1 Transgenic Mouse Model

Blood ◽

10.1182/blood.v124.21.283.283 ◽

2014 ◽

Vol 124 (21) ◽

pp. 283-283

Author(s):

Jesus Duque-Afonso ◽

Jue Feng ◽

Florian Scherer ◽

Zhong Wang ◽

Michael L. Cleary

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Disease Free Survival ◽

Leukemia Cells ◽

Mouse Strains ◽

Driver Gene ◽

Hematopoietic Stem

Abstract Pediatric acute lymphoblastic leukemia (ALL) represents a collection of orphan diseases that are defined by their genomic abnormalities. Their genetic diversity and low prevalence serve as major barriers to investigations of their molecular pathogenesis and translational biology. To address this, we have engineered novel mouse strains that conditionally activate and express E2a-PBX1, the fusion oncogene derived from chromosomal translocation t(1;19), present in 5-7% of pediatric ALL. Somatic activation of the oncogene is accomplished by Cre-recombinase expressed under the control of specific B-lineage promoters CD19 or Mb1, or in hematopoietic stem cells using the Mx1 interferon-inducible promoter. The three mouse strains show similar pre-leukemic and leukemic phenotypes. At the time of disease, mice exhibit leukocytosis, anemia and thrombocytopenia as well as lymphadenopathy and hepatosplenomegaly and infiltration of several organs including kidney, lung and central nervous system. Leukemia cell phenotypes (CD117+, CD19+, CD43+, CD45+, CD25+, sIgM-, Bp-1+, CD24+, CD127+, CD79a+ and TdT+) correspond to the phenotypic fraction B-C’ (or pro-B/large pre-B II stage, Basel nomenclature) that is very similar to human E2a-PBX1+ pre-B-ALL. Hence, we detected productive VDJ rearrangements and cytoplasmic heavy chain in 12.5 % of cases, a characteristic of human E2a-PBX1 leukemias. Leukemia incidence varies from 5-50% depending on the Cre driver gene and the median latency is about 10 months in E2a.PBX1/Mb1.Cre and Mx1.Cre lines, suggesting the need for secondary mutations. Whole exome sequencing detected secondary genetic aberrations, which were validated in a larger cohort of leukemias. Spontaneous deletions of Pax5, which are present in ~45% of pediatric ALLs with E2a-PBX1 gene fusions, were found in about 30 % of mouse E2a-PBX1 leukemias. Conditional deletion of Pax5 and E2a-PBX1 expression expanded progenitor B cell subpopulations in healthy 3-months old preleukemic mice. Consequently, Pax5 haplo-insufficiency in mice cooperates with E2a-PBX1 increasing the penetrance and shortening the latency of leukemia, providing the first evidence for cooperative oncogenic effects of Pax5 haplo-insufficiency. Tumor suppressor genes as Trp53 and Cdkn2a/b were inactivated by secondary mutations and deletions, respectively. Additionally, secondary recurrent activating mutations were detected in key signaling pathways such as Ras/Mapk and Jak/Stat on which the leukemia cells are strongly dependent. Furthermore, leukemia cells displayed higher basal levels of phosphorylated pSTAT5 and pAKT, pERK1/2, and were hyper-sensitive to stimulation with IL-7 and thymic stromal lymphopoietin (TSLP) as seen by induction of pSTAT5 and supported growth in colony-forming assays. The JAK1/2 inhibitor ruxolitinib blocked the induction of pSTAT5 by IL-7 and TSLP, inhibited colony formation in vitro, and increased disease-free survival after in vivo treatment. Human E2a-PBX1 primary cells and cell lines showed hypersensitivity to IL-7/pSTAT5 activation compared to other ALL karyotypes and pre-treatment with ruxolitinib blocked induction of pSTAT5 by IL-7. In summary, we have developed conditional transgenic E2a-PBX1 mouse models that consistently develop leukemias that resemble human pre-B-ALL carrying the t(1;19) and identified key cooperating oncogenic pathways. This model provides experimental validation of the multistep pathogenesis for a subset of ALL previously inferred from genomic analyses and provides a platform for comparative mechanistic and preclinical studies. Disclosures No relevant conflicts of interest to declare.

Download Full-text

In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling

Blood ◽

10.1182/blood.v118.21.758.758 ◽

2011 ◽

Vol 118 (21) ◽

pp. 758-758

Author(s):

◽

Fatima Al-Shahrour ◽

Kimberly A. Hartwell ◽

Lisa P Chu ◽

Jaras Marcus ◽

...

Keyword(s):

Rna Interference ◽

Cell Growth ◽

Leukemia Cell ◽

Leukemia Cells ◽

Growth And Survival ◽

Integrin Beta 3 ◽

Shrna Screen ◽

Primary Leukemia

Abstract Abstract 758 Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3). In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment. We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures: Armstrong: Epizyme: Consultancy.

Download Full-text

Novel CD19/CD22 Bicistronic Chimeric Antigen Receptors Outperform Single or Bivalent Cars in Eradicating CD19+CD22+, CD19-, and CD22- Pre-B Leukemia

Blood ◽

10.1182/blood.v130.suppl_1.810.810 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 810-810 ◽

Cited By ~ 3

Author(s):

Haiying Qin ◽

Sang M Nguyen ◽

Sneha Ramakrishna ◽

Samiksha Tarun ◽

Lila Yang ◽

...

Keyword(s):

T Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Single Chain ◽

Dual Targeting ◽

Single Antigen ◽

Therapeutic Function

Abstract Treatment of pre-B cell acute lymphoblastic leukemia (ALL) using chimeric antigen receptor expressing T cells (CART) targeting CD19 have demonstrated impressive clinical results in children and young adults with up to 70-90% complete remission rate in multiple clinical trials. However, about 30% of patients relapse due to loss of the targeted epitope on CD19 or CART failure. Our CD22-targeted CAR trial has generated promising results in relapsed/refractory ALL, including CD19 antigen negative ALL, but relapse associated with decreased CD22 site density has occurred. Thus, developing strategies to prevent relapses due to changes in antigen expression have the potential to increase the likelihood of durable remissions. In addition, dual targeting of both CD19 and CD22 on pre-B ALL may be synergistic compared to targeting a single antigen, a potential approach to improve efficacy in patients with heterogeneous expression of CD19 and CD22 on leukemic blasts. We describe the systematic development and comparison of the structure and therapeutic function of three different types (over 15 different constructs) of novel CARs targeting both CD19 and CD22: (1) Bivalent Tandem CAR, (2) Bivalent Loop CAR, and (3) Bicistronic CAR. These dual CARs were assembled using CD19- and CD22-binding single chain fragment variable (scFv) regions derived from clinically validated single antigen targeted CARs. They are structurally different in design: both tandem and loop CARs have the CD19 and CD22 scFv covalently linked in the same CAR in different orders, whereas, bicistronic CARs have 2 complete CAR constructs connected with a cleavable linker. The surface expression on the transduced T cell of the CD19/CD22 dual CARs was detected with CD22 Fc and anti-idiotype of CD19 and compared to single CD19 or CD22 CARs. Activities of dual CARs to either CD19 or CD22 were evaluated in vitro with cytotoxicity assays or killing assays against K562 cells expressing either CD19 or CD22 or both antigens and also tested against a leukemia CD19+/CD22+ cell line, NALM6, and NALM6 with CRISPER/CAS9 knockout of CD19 or CD22 or both antigens. Therapeutic function of the top candidates of the dual CARs was then validated in vivo against these NALM6 leukemia lines. Some of these dual CARs were also further tested against patient-derived xenografts. Finally, we tested the dual targeting CARs in an artificial relapse model in which mice were co-injected with a mix of CD19 knockout and CD22 knockout NALM6 leukemia lines. From these studies, we established that the order of the scFv, size of the linker, type of leader sequence, and co-stimulatory domain in the CAR constructs all impact the efficacy of the dual targeting CARs. Tandem, Loop, and Bicistronic CARs all demonstrate some levels of in vitro and in vivo activities, but the bicistronic CAR was most effective at clearing leukemia and preventing relapse. In the CD19+/CD22+ NALM6 model, bicistronic CAR treated mice remain disease free while CD19 CAR or CD22 CAR treated mice already died or relapsed on day 27. In the relapse model, as expected, CD19 or CD22 single CAR T cell treatment resulted in progression of the corresponding antigen-negative NALM6. Treatment with dual targeted bicistronic CARs resulted in clearance of both CD19 and CD22 negative ALL with durable remission. In summary, we described novel CD19/CD22 dual targeting CARs with robust pre-clinical activity against pre-B cell ALL, and validated this approach in the prevention of resistance to single-antigen targeted CARs in preclinical models. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Curcumin and Its Analogue Induce Apoptosis in Leukemia Cells and Have Additive Effects with Bortezomib in Cellular and Xenograft Models

BioMed Research International ◽

10.1155/2015/968981 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 16

Author(s):

L. I. Nagy ◽

L. Z. Fehér ◽

G. J. Szebeni ◽

M. Gyuris ◽

P. Sipos ◽

...

Keyword(s):

Leukemia Cell ◽

Leukemic Cells ◽

Leukemia Cell Line ◽

Leukemia Cells ◽

Great Promise ◽

Human Leukemia ◽

Human Leukemia Cell Line ◽

Apoptotic Effects

Combination therapy of bortezomib with other chemotherapeutics is an emerging treatment strategy. Since both curcumin and bortezomib inhibit NF-κB, we tested the effects of their combination on leukemia cells. To improve potency, a novel Mannich-type curcumin derivative, C-150, was synthesized. Curcumin and its analogue showed potent antiproliferative and apoptotic effects on the human leukemia cell line, HL60, with different potency but similar additive properties with bortezomib. Additive antiproliferative effects were correlated well with LPS-induced NF-κB inhibition results. Gene expression data on cell cycle and apoptosis related genes, obtained by high-throughput QPCR, showed that curcumin and its analogue act through similar signaling pathways. In correlation with in vitro results similar additive effect could be obsereved in SCID mice inoculated systemically with HL60 cells. C-150 in a liposomal formulation given intravenously in combination with bortezomib was more efficient than either of the drugs alone. As our novel curcumin analogue exerted anticancer effects in leukemic cells at submicromolar concentration in vitro and at 3 mg/kg dose in vivo, which was potentiated by bortezomib, it holds a great promise as a future therapeutic agent in the treatment of leukemia alone or in combination.

Download Full-text

Transient Responses to Notch and Tlx1/Hox11 Inhibition In T-Cell Acute Lymphoblastic Leukemia/Lymphoma.

Blood ◽

10.1182/blood.v116.21.1026.1026 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1026-1026

Author(s):

Erica A. Lehotzky ◽

Mark Y. Chiang

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Line ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Ectopic Expression ◽

Leukemia Cells ◽

Inhibitory Effects ◽

Notch Inhibition

Abstract Abstract 1026 Despite numerous advances in the past few decades, treatment of acute lymphoblastic leukemia/lymphoma (ALL) remains a common and considerable challenge. Further efforts to define the molecular lesions that drive ALL are needed to improve clinical management. The Hox subfamily of T-cell ALL (T-ALL) represents 30–40% of pediatric and adult cases. TLX1/HOX11 is the prototypical member of the Hox group. To generate a resource for developing targeted therapies for Hox T-ALLs, we developed a doxycycline-regulated mouse model of Tlx1-initiated T-ALL. Dysregulated thymic expression of Tlx1 induces T-ALL after ∼5-7 months with penetrance of 15–60%. The lymphoblasts are arrested at the early CD4+/CD8+/CD24hi stage of T-cell development, similar to human T-ALLs of the TLX1 subtype. Spontaneous activation of the Notch1 oncogene occurred in the tumors. In about two-thirds of samples, Notch was activated through acquired mutations in the heterodimerization and PEST domains that resemble the Notch1 mutations found in human patients. Inhibition of Notch signaling with g-secretase inhibitors completely abrogated cell line growth and induced apoptosis. Notch inhibition also transiently delayed leukemia progression by ∼17 days in vivo. In contrast, suppression of Tlx1 expression had more moderate inhibitory effects on cell line growth in vitro. However, suppression of Tlx1 expression in transgenic mice transiently delayed leukemia progression by ∼11 days. Tlx1 suppression had the strongest inhibitory effects on expression of CCR7 and lymph node size. These effects were fully reversed with ectopic expression of Tlx1. These data show that Tlx1 can convert normal thymocytes into leukemia cells, but the leukemia cells are not fully dependent on continued Tlx1 expression. The leukemia cells recruit secondary factors and pathways such as Notch and c-Myc to sustain growth and survival. Our study highlights a strong resiliency of T-ALL cells to both Tlx1 and Notch inhibition. Our study has important implications for targeting these pathways for the treatment of T-ALL. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Quantitative Phosphoproteomics Identified a New Syk-Lyn-Axl Signalling Pathway Involved In Resistance to Nilotinib In Chronic Myeloid Leukemia Cells.

Blood ◽

10.1182/blood.v116.21.3376.3376 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3376-3376

Author(s):

Romain Gioia ◽

Cedric Leroy ◽

Claire Drullion ◽

Valérie Lagarde ◽

Serge Roche ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Critical Role ◽

Dependent Manner ◽

Stable Isotope Labelling ◽

Resistant Cells

Abstract Abstract 3376 Nilotinib has been developed to overcome resistance to imatinib, the first line treatment of chronic myeloid leukemia (CML). To anticipate resistance to nilotinib, we generate nilotinib resistant CML cell lines in vitro to characterize mechanisms and signaling pathways that may contribute to resistance. Among the different mechanisms of resistance identified, the overexpression of the Src-kinase Lyn was involved in resistance both in vitro, in a K562 cell line (K562-rn), and in vivo, in nilotinib-resistant CML patients. To characterize how Lyn mediates resistance, we performed a phosphoproteomic study using SILAC (Stable Isotope Labelling with Amino acid in Cell culture). Quantification and identification of phosphotyrosine proteins in the nilotinib resistant cells point out two tyrosine kinases, the spleen tyrosine kinase Syk and the UFO receptor Axl. The two tyrosine kinase Syk and Axl interact with Lyn as seen by coimmunopreciptation. Syk is phosphorylated on tyrosine 323 and 525/526 in Lyn dependent manner in nilotinib resistant cells. The inhibition of Syk tyrosine kinase by R406 or BAY31-6606 restores sensitivity to nilotinib in K562-rn cells. In parallel, the inhibition of Syk expression by ShRNA in K562-rn cells abolishes Lyn and Axl phosphorylation and then interaction between Lyn and Axl leading to a full restoration of nilotinib efficacy. In the opposite, the coexpression of Lyn and Syk in nilotinib sensitive K562 cells induced resistance to nilotinib whereas a Syk kinase dead mutant did not. These results highlight for the first time the critical role of Syk in resistance to tyrosine kinase inhibitors in CML disease emphasizing the therapeutic targeting of this tyrosine kinase. Moreover, Axl, which is already a target in solid tumor, will be also an interesting pathway to target in CML. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

MHC Class II and T Cell Receptor Signals Are Dispensable for IL-2-Induced Regulatory T Cell Proliferation In Vivo

Blood ◽

10.1182/blood.v118.21.2181.2181 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2181-2181

Author(s):

Tao Zou ◽

Atsushi Satake ◽

Jonathan Maltzman ◽

Taku Kambayashi

Keyword(s):

T Cell ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Adaptor Protein ◽

Class Ii ◽

Sh2 Domain ◽

Tcr Signaling ◽

Mhc Ii

Abstract Abstract 2181 Regulatory T cells (Tregs) protect the host from autoimmunity and inappropriate immune activation. Thus, to ensure immune tolerance in the steady state, an adequate number of peripheral Tregs must be constantly maintained. Prior work has suggested that major histocompatibility class II (MHC II) and interleukin-2 (IL-2) are both necessary to maintain peripheral Treg homeostasis and proliferation in vivo. However, we have recently reported that Treg proliferation may not strictly depend on MHC II, as the provision of IL-2 was sufficient to drive proliferation of Tregs in an MHC II-independent manner in vitro, as long as the Tregs interacted with dendritic cells (DC)s. Here, extending our previous in vitro observations, we tested the dependence of Treg proliferation on IL-2, DCs, and TCR signaling in vivo. Proliferation of adoptively transferred Tregs was detected in wildtype (WT) mice. This proliferation was markedly enhanced when the mice were injected with IL-2 immune complexes (IC)s but not when the IL-2 IC-injected mice lacked DCs, suggesting that IL-2-induced Treg proliferation was dependent on DCs in vivo. As previously reported, adoptively transferred Tregs did not proliferate in MHC II-deficient hosts. However, the injection of IL-2 ICs into these mice induced Treg proliferation comparable to those transferred into IL-2 IC-injected WT mice, suggesting that IL-2 signaling by Tregs obviated the need of MHC II for their proliferation. Furthermore, while the ablation of TCR signaling by conditional deletion of the adaptor protein SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) rendered Tregs unable to proliferate by themselves, IL-2 IC treatment partially rescued this deficiency. We next examined the signaling pathways involved in Treg proliferation downstream of the IL-2 receptor. Despite the importance of the Stat5 pathway in IL-2 receptor signaling during Treg development in the thymus, activation of Stat5b alone was insufficient to rescue proliferation of SLP-76-deficient Tregs, indicating that alternative pathways must also be activated for Treg proliferation. Additional studies investigating the role of other signaling molecules downstream of the IL-2 receptor are currently underway. In summary, we have demonstrated for the first time that Tregs do not require TCR signaling through interaction with MHC II for their proliferation in vivo. We propose that this MHC II-independent mode of Treg proliferation allows Tregs with multiple antigen specificities to proliferate, which ensures that a diverse TCR repertoire is continuously maintained in the Treg pool. Furthermore, we believe that exploitation of these pathways may be therapeutically beneficial in autoimmunity and in transplantation. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

ARC Is Regulated by MAPK and PI3K and Confers Drug Resistance and Survival Advantage to AML in Vitro and in Vivo

Blood ◽

10.1182/blood.v120.21.893.893 ◽

2012 ◽

Vol 120 (21) ◽

pp. 893-893

Author(s):

Po Yee Mak ◽

Duncan H Mak ◽

Yuexi Shi ◽

Vivian Ruvolo ◽

Rodrigo Jacamo ◽

...

Keyword(s):

Bone Marrow ◽

Leukemia Cell ◽

Leukemia Cells ◽

Apoptosis Resistance ◽

Control Mscs ◽

Extrinsic Apoptosis ◽

Induced Apoptosis ◽

And Control

Abstract Abstract 893 ARC (Apoptosis repressor with caspase recruitment domain) is a unique antiapoptotic protein that has been shown to suppress the activation of both intrinsic and extrinsic apoptosis. We previously reported that ARC is one of the most potent adverse prognostic factors in AML and that high ARC protein expression predicted shorter survival and poor clinical outcome in patients with AML (Carter BZ et al., Blood 2011). Here we report how ARC is regulated and its role in inhibition of AML apoptosis and in cell survival. We provide evidence that ARC expression is regulated by MAPK and PI3K signaling. Inhibition of MAPK and PI3K pathways decreased ARC mRNA and protein levels in AML cells. ARC expression in AML cells is upregulated in co-cultures with bone marrow-derived mesenchymal stromal cells (MSCs) and the upregulation is suppressed in the presence of MAPK or PI3K inhibitors. To investigate the role of ARC in apoptosis resistance in AML, we generated stable ARC overexpressing (O/E) KG-1 and stable ARC knock down (K/D) OCI-AML3 and Molm13 cells and treated them with Ara-C and agents selectively inducing intrinsic (ABT-737) or extrinsic (TRAIL) apoptosis. We found that ARC O/E cells are more resistant and ARC K/D cells more sensitive to Ara-C, ABT-737, and TRAIL-induced apoptosis: EC50s of Ara-C, ABT-737, or TRAIL treatment at 48 hours for ARC O/E KG-1 and control cells were 1.5 ± 0.1 μM vs. 83.5 ± 4.6 nM, 2.2 ± 0.2 μM vs. 60.2 ± 3.1 nM, or 0.97 ± 0.03 μg/mL vs. 0.17 ± 0.08 μg/mL, respectively and for ARC K/D OCI-AML3 and control cells were 0.33 ± 0.02 μM vs. 3.4 ± 0.2 μM, 0.24 ± 0.01 μM vs. 1.3 ± 0.1 μM, or 0.13 ± 0.09 μg/mL vs. 0.36 ± 0.03 μg/mL, respectively. Bone marrow microenvironment is known to play critical roles in AML disease progression and in protecting leukemia cells from various therapeutic agent-induced apoptosis. Leukemia cells were co-cultured with MSCs in vitro study to mimic the in vivo condition. ARC was found to be highly expressed in MSCs and stable ARC K/D MSCs were generated. AML cell lines and primary patient samples were co-cultured with ARC K/D or control MSCs and treated with Ara-C, ABT-737, or TRAIL. Interestingly, ARC K/D MSCs lost their protective activity for leukemia cells treated with these agents. EC50s for OCI-AML3 cells co-cultured with ARC K/D or control MSCs for 48 hours treated with Ara-C, ABT-737, or TRAIL were 1.0 ± 0.04 μM vs. 4.5 ± 0.2 μM, 0.15 ± 0.06 μM vs. 0.53 ± 0.02 μM, or 1.4 ± 0.8 μg/mL vs. 8.1 ± 0.3 μg/mL, respectively. In addition, ARC O/E KG-1 cells grew faster and ARC K/D OCI-AML3 and Molm13 cells and ARC K/D MSCs grew slower than their respective controls. We then injected KG-1 cells into mice and found that NOD-SCID mice harboring ARC O/E KG-1 had significantly shorter survival than mice injected with the vector control KG-1 (median 84 vs. 111 days) as shown in the figure. Collectively, results demonstrate that ARC plays critical roles in AML. ARC is regulated by MSCs through various signaling pathways in AML cells, protects leukemia cells from apoptosis induced by chemotherapy and by agents selectively inducing intrinsic and extrinsic apoptosis. ARC regulates leukemia cell growth in vitro and in vivo. The results suggest that ARC is a potential target for AML therapy. In addition, targeting ARC in MSCs suppresses microenvironmental protection of AML cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text