Yao-Te Hsieh

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1–110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/β- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

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

Bone 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.

Integrated microfluidic and imaging platform for a kinase activity radioassay to analyze minute patient cancer samples

Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric (32)P-ATP-labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL-positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples.

Effects of the small-molecule inhibitor of integrin α4, TBC3486, on pre-B-ALL cells

The treatment of patients with chemotherapy-resistant leukemia remains a challenge. A role of the microenvironment for drug resistance of leukemia cells has been proposed.1 We have identified the adhesion molecule integrin α4 as a central mediator of drug resistance of pre-B-cell acute lymphoblastic leukemia (ALL).2 We thus demonstrated that chemotherapy-resistant pre-B-ALL cells can be eradicated in a xenograft model by concurrent blockade of α4 using natalizumab, a humanized anti-α4 antibody in clinical use against multiple sclerosis,3 and Crohns Disease.2 Here, we extended our studies to an alternative α4 inhibitor, the non-peptidic small molecule TBC3486. Previous in vitro assays and molecular modeling studies indicated that TBC3486 behaves as a ligand mimetic, competing with VCAM-1 for the MIDAS site of integrin α4.4 As such, the compound has shown efficacy in integrin α4-dependent models of inflammatory and autoimmune disease4 and has shown efficacy in mice with autoimmune encephalomyelitis, a model for multiple sclerosis.5 As opposed to natalizumab, which will inhibit both members of the α4 integrin family, α4β1 and α4β7, TBC3486 is 200-fold more potent in inhibiting α4β1 than α4β7. In addition, it is completely inactive against all other integrins tested, including members of the β2, β3 as well as other members of the β1 family of integrins.4 The potential usefulness of this novel inhibitor for pre-B-ALL treatment was tested in our established in vitro and in vivo assays.2, 4 We evaluated the effect of TBC3486 on de-adhesion of patient-derived ALL cells (LAX7R) using established adhesion assays. As a control for our studies, a close structural analog was used that lacks activity toward α4β1 integrin (THI0012). After activating LAX7R cells with 1 mM Mn2+, leukemia cells were co-cultured with the murine stromal cell line OP9.6, 7 Subsequently, LAX7R cells were treated with different doses of TBC3486 (5, 10 and 25 μM) and its control, THI0012 (5, 10 and 25 μM), for 4 days. TBC3486 dose-dependently inhibited adhesion of ALL cells (Figure 1a), albeit the adhesion was not completely blocked. The dose of 25 μM was selected for subsequent studies. The concentrations of compound required for inhibition in these assays are higher than previously reported.4 This is due to the fact that TB3486 is highly protein bound in the presence of 20% serum (used in these assays), which significantly reduces the amount of free compound available to bind to the integrin receptor. Next, we determined whether TBC3486 decreases binding of three xenograft cells derived from primary pre-B-ALL cases (LAX7R, ICN3 and SFO3) to the counter-receptor of α4 integrin, human VCAM-1. Adhesion assays were performed as previously described4, 8, 9 by culturing ALL samples treated with TBC3486 (25 μM) or THI0012 (25 μM) on hVCAM-1-coated plates for 2 days. Compared with the control group, TBC3486-treated ALL cells showed significantly less adhesion to hVCAM-1 (Figures 1c, e and g); however, the adhesion was not completely blocked. CD49d (MFI) is expressed with higher intensity in LAX7R compared with the other two samples (ICN3 and SFO3) (data not shown), which may explain why TBC3486 blocked a larger percentage of LAX7R adhesion to VCAM-1. In addition to blocking cell adhesion, TBC3486 treatment also specifically targeted the expression of integrin α4, but not integrin α5 and α6 (Figure 1b). The treatment with TBC3486 did not affect cell viability in all three cases (Figures 1d, f and h) compared with the THI0012 control. Taken together, TBC3486 leads to the partial de-adhesion of pre-B-ALL cells from its counter-receptor VCAM-1 under the conditions described.

AMD3100 sensitizes acute lymphoblastic leukemia cells to chemotherapy in vivo

Despite substantial advances in the treatment of acute lymphoblastic leukemia (ALL) during the past four decades, long-term survival remains at approximately 80% for children and 40% for adults. The lack of efficacy of treatment can be partly attributed to the protection provided to the leukemia cells by the bone marrow microenvironment. SDF-1α (CXCL12) is a cytokine produced by bone marrow stromal cells that stimulates the growth of pre-B cells. Its receptor, CXCR4, is expressed on mature and precursor hematopoietic cells. The CXCL12–CXCR4 interaction was shown to be essential for the earliest stages of B-cell development and is also involved in retaining pre-B cells in the bone marrow. Original interest in CXCR4 from a therapeutic point of view was focused on its role as a co-receptor for human immunodeficiency virus, but drugs that block CXCR4, such as AMD3100, can also be used for hematopoietic stem cell mobilization.1 Juarez et al.2, 3 reported that CXCR4 receptor-binding drugs inhibit CXCR4-mediated functions of pre-B ALL cells in vitro and can mobilize transplanted ALL cells into the circulation of mice. However, the effect of combined CXCR4 inhibition with chemotherapy on ALL remains to be determined. We previously showed that AMD3100 is able to block protection provided by a stromal layer to mouse transgenic Bcr/Abl P190 ALL cells treated with Imatinib.4 To test whether these results can be extended in vivo, we performed fluorescence-activated cell sorting analysis on the peripheral blood of leukemic P190 Bcr/Abl ALL (8093)-transplanted mice before and 2 h after intraperitoneal injection with saline or AMD3100, as it has been shown that a single bolus of AMD3100 can mobilize progenitor cells.1 Interestingly, there was a marked increase in circulating leukemic CD45.2+ (8093 ALL) cells after injection of AMD3100 (Figure 1a) but not in mobilized CD45.1+ C57BL/6 cells. This lack of effect on endogenous CD45+ cells may be caused by the suppression of normal hematopoiesis by the presence of large numbers of malignant lymphoblasts in the bone marrow at this stage. Using the same model, we also examined whether AMD3100 is able to repeatedly mobilize ALL cells in leukemic mice over a longer period of time. As shown in Figure 1b, AMD3100 was able to mobilize a similarly large number of cells into the circulation at days 1 and 10, indicating a sustained mobilizing effect of this drug. These results are consistent with those found in murine transplant models of acute promyelocytic leukemia and multiple myeloma .5, 6 To investigate whether their mobilization into the peripheral blood will make ALL cells more vulnerable to drug treatment, we next tested the combination effect of AMD3100 and nilotinib (AMN107) in vivo. Leukemia cells were allowed to proliferate and generate a substantial tumor burden before the start of treatment with phosphate-buffered saline, nilotinib, AMD3100, or a combination of nilotinib and AMD3100 (Figure 1c). As reported previously,7 nilotinib treatment prolonged the survival of mice. AMD3100 treatment alone had no significant beneficial or detrimental effect on survival (Figure 1c). Interestingly, combination therapy using AMD3100 and nilotinib led to significantly prolonged survival in this murine leukemia model compared with treatment with only nilotinib (P<0.05). Figure 1 Combination therapy using AMD3100 and nilotinib prolongs survival of murine ALL transplant recipients. C57Bl mice transplanted with 104 Bcr/Abl P190 ALL 8093 cells were allowed to develop full leukemia within 11–17 days. (a) Fold increase 2 h ... We verified that the AMD3100-mobilized cells included ALL cells by engrafting non-obese diabetic/severe combined immunodeficient mice with the human pre-B ALL cell line 697. As shown in Figure 2a, the increased white blood cells at 2 h after injection with AMD3100 (top) correlated with a significant increase in the peripheral blood of cells expressing human CD19 (bottom), a marker that is highly expressed in this ALL (>99%, data not shown). We next tested the combined treatment of chemotherapy and AMD3100 in vivo using primary human US7R, a Philadelphia chromosome-negative ALL. At 12 days after transplant with US7R, mice were started on treatment with phosphate-buffered saline, AMD3100, VDL (Vincristine, Dexamethasone, -asparaginase) or AMD3100 plus VDL. The untreated control group died rapidly 27 days post-leukemia injection (Figure 2b). Interestingly, mice treated with VDL plus AMD3100 (MST=61.5 days) survived significantly longer compared with those treated with VDL alone (MST=54 days; P=0.015) or AMD3100 alone (MST=27 days; P=0.0022). Treatment with AMD3100 appeared to be well tolerated, as indicated by continuous weight gain in the treatment groups (data not shown). Figure 2 Preclinical evaluation of AMD3100 as an adjuvant treatment for human drug-resistant ALL. (a) Mobilization of human ALL cells. At 12 days after transplant of human pre-B ALL 697 (6 × 106 cells/mouse) NOD/SCID/IL2Rγ−/− mice ... The concept of using mobilizing agents to bring ALL cells into the circulation in which they can be more effectively treated with other drugs had not been tested in vivo, although Juarez et al.2 did show that AMD3100 enhanced the cytotoxic and anti-proliferative effects of vincristine and dexamethasone in pre-B ALL cells in culture. In AML, AMD3465, a compound related to AMD3100, enhanced the anti-leukemic effects of chemotherapy and sorafenib in mouse transplant models.8 Use of AMD3100 with Ara-C or with bortezomib in acute promyelocytic leukemia or multiple myeloma also showed that combination treatment sensitized these cancer cells to the therapeutic drug in mouse models.5, 6 Moreover, there are currently ongoing Phase clinical I/II trials (http://clinicaltrials.gov identifier: NCT00512252) for the study of AMD3100 in relapsed or refractory acute myelogenous leukemia in combination with chemotherapy with mitoxantrone, etoposide and cytarabine. In view of the fact, that our studies were performed in models of very advanced primary ALL, in which the animals were allowed to accumulate a substantial tumor burden before treatment was initiated, combined with the fact that the human US7R cells are largely unresponsive to the therapeutic drug combination VDL (results not shown); the effect of AMD3100 combined with a second drug can be regarded as very promising. Thus, clinical trials that test the usefulness of combination treatment with CXCR4 antagonists for therapy of relapsed or high-risk ALL appear to be warranted.