The effect of the dual Src/Abl kinase inhibitor AZD0530 on Philadelphia positive leukaemia cell lines

Background Imatinib mesylate, a selective inhibitor of Abl tyrosine kinase, is efficacious in treating chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL). However, most advanced-phase CML and Ph+ ALL patients relapse on Imatinib therapy. Several mechanisms of refractoriness have been reported, including the activation of the Src-family kinases (SFK). Here, we investigated the biological effect of the new specific dual Src/Abl kinase inhibitor AZD0530 on Ph+ leukaemic cells. Methods Cell lines used included BV173 (CML in myeloid blast crisis), SEM t(4;11), Ba/F3 (IL-3 dependent murine pro B), p185Bcr-Abl infected Ba/F3 cells, p185Bcr-Abl mutant infected Ba/F3 cells, SupB15 (Ph+ ALL) and Imatinib resistant SupB15 (RTSupB15) (Ph+ ALL) cells. Cells were exposed to AZD0530 and Imatinib. Cell proliferation, apoptosis, survival and signalling pathways were assessed by dye exclusion, flow cytometry and Western blotting respectively. Results AZD0530 specifically inhibited the growth of, and induced apoptosis in CML and Ph+ ALL cells in a dose dependent manner, but showed only marginal effects on Ph- ALL cells. Resistance to Imatinib due to the mutation Y253F in p185Bcr-Abl was overcome by AZD0530. Combination of AZD0530 and Imatinib showed an additive inhibitory effect on the proliferation of CML BV173 cells but not on Ph+ ALL SupB15 cells. An ongoing transphosphorylation was demonstrated between SFKs and Bcr-Abl. AZD0530 significantly down-regulated the activation of survival signalling pathways in Ph+ cells, resistant or sensitive to Imatinib, with the exception of the RTSupB15. Conclusion Our results indicate that AZD0530 targets both Src and Bcr-Abl kinase activity and reduces the leukaemic maintenance by Bcr-Abl

Different Roles of Two Autotaxin Isoforms in Proliferation, Migration and Adhesion in the Non-Mutational Tyrosine Kinase Inhibitor Resistant Acute Lymphoblastic Leukemia Cell Line SupB15.

Abstract The Bcr-Abl oncogene is present in 30–40% of adult patients with acute lymphoblastic leukemia (ALL). The Abl kinase inhibitor imatinib-based therapy has become standard for this subset ALL. Acquired resistance to imatinib occurs frequently and is associated with mutations in the tyrosine kinase domain (TKD) approximately in about 80% of patients. In contrast, TKD mutations are uncommon in primary imatinib resistance which appears to be multifactorial, although the underlying mechanisms have been incompletely elucidated. We have established a Ph+ cell line for the analysis of non-mutational resistance mechanisms of imatinib resistance: SupB15RT, a Bcr-Abl expressing lymphoblastic cell line derived from SupB15WT cell line by gradually increasing the exposure to imatinib. SupB15RT shows cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. We have shown that several commonly implicated mechanisms of imatinib resistance do not play a role in conferring the imatinib resistance in SupB15RT cells. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affymetrix- Microarrays, we identified 29 differentially regulated genes. Autotaxin (ATX) is one of the most highly up-regulated genes in imatinib resistant SupB15RT cells, and suggested a contribution to imatinib resistance. ATX is an exo-enzyme (pyrophosphophatase/phosphodiesterase). It plays a role in tumor progression and migration as a tumor cell autocrine motilty factor in various solid tumor cell types. ATX is involved in the synthesis of the signaling molecule, lysophosphatidic acid (LPA) which promotes survival and motility. It was the aim of this study to determine whereas ATX plays a functional role for imatinib resistance in Ph+ ALL. Using RT-PCR we demonstrated that 2 isoforms of ATX are expressed in SupB15RT cells: ATXshort and ATXlong. ATXlong (863 aa) contains highly basic insertion in the catalytic domain (52 residues). We retroviraly transfected BaF3 cells with p185 and/or ATXshort or ATXlong to analyze its influence on growth, adhesion and migration in mouse cell model. In comparison to wild type BaF3 cells the proliferation of BaF3 cells expressing ATXshort is enhanced (1,5-fold), whereas ATXlong expressing BaF3 cells showed no difference in proliferation in comparison to Mock infected cells. The proliferation of p185 expressing BaF3 cells co-expressing ATXshort or ATXlong is not inhibited by the treatment with 1μM imatinib after 3 days in contrast to p185 expressing BaF3 cells. In adhesion experiments, BaF3 cells expressing ATXshort showed a higher attachment independent of p185 expression. We also performed migration experiments using transwell assays. These assays showed more migration with cells co-expressing p185 and ATXlong compared to p185 alone. This is in agreement with our results for SupB15RT vs. SupB15WT with a 3-fold migration increase of SupB15RT. Application of 10% fetal calf serum (FCS) in migration experiments resulted in a 1,5-fold higher migration of the ATXlong expressing BaF3 cells compared to culture without FCS. One explanation for this finding may be that FCS contains lysophosphatidic choline (LPC) which is converted to LPA by ATX. Although expression of both 2 isoforms of ATX is important for the increased proliferation, it seems that the 2 isoforms have different cellular functions in Ph+ lymphoblastic cells. ATXshort seems to enhance adhesion whereas ATXlong plays an important role in motility. Taken together our results indicate a role for ATX in TK- inhibitor resistant SupB15RT cells through LPA signaling via LPA receptors. The ratio between ATXshort and ATXlong probably is important for the intracellular signaling and has to be explored.</jats:p

A Role of Autotaxin in Non-Mutational Resistance in Imatinib-Resistant Acute Lymphoblastic Leukemia (ALL) Cell Line SupB15RT.

Abstract The Bcr-Abl oncogene is present in 30-40% of adult patients with acute lymphoblastic leukemia (ALL). Therapy with imatinib has become standard for Ph+ ALL but resistance to the tyrosine kinase inhibitor occurs for the majority of patients. In about 80% of patients with acquired resistance mutations in the tyrosine kinase domain (TKD) have been found. In contrast, primary resistance to imatinib appears to be multifactorial and precise mechanisms have been incompletely elucidated. We have established an imatinib-resistant cell line (SupB15RT) which was derived from the previously well characterized SupB15 cell line (SupB15WT) by gradually increasing the exposure to imatinib. We found that several commonly implicated mechanisms of imatinib resistance, i.e. Bcr-Abl gene amplification, point mutations in the TKD, Bcr-Abl overexpression, up-regulation of multidrug resistance gene proteins or ineffective inhibition of Bcr-Abl tyrosine phosphorylation do not play a role in conferring the imatinib-resistant phenotype in SupB15RT cells. Thus, the SupB15RT cells represent a suitable model for the analysis of resistance mechanisms in Ph+ ALL with primary imatinib resistance. Interestingly, SupB15RT cells show cross-resistance to the second generation Abl kinase inhibitors Nilotinib and Dasatinib. Analysis of signal transduction pathways downstream of Bcr-Abl revealed that imatinib exposure was not associated with down-regulation of pSTAT-5 and pErk in the imatinib-resistant SupB15RT cells, in contrast to SupB15WT. Phosphorylation of Akt was inhibited by 0.5μM imatinib in SupB15WT cells, whereas imatinib in concentrations up to 5μM failed to suppress Akt phosphorylation in SupB15RT cells, indicating constitutive activation of Akt kinase during imatinib treatment. By comparative gene expression analysis of SupB15WT vs. SupB15RT cells using Affimetrix-Microarrays, we identified 29 differentially regulated (at least 3-fold) genes. One of the most highly up-regulated genes in imatinib-resistant SupB15RT cells was Autotaxin (ATX), a nucleotide pyrophosphatase/ phosphodiesterase 2. This exo-enzyme was originally identified as a tumor cell autocrine motility factor, which is involved in tumor progression and migration in various tumor cell types. ATX is a lysophospholipase D which is involved in the synthesis of lysophosphatidic acid (LPA), a signaling molecule that promotes survival, growth, differentiation, and motility. We investigated if LPA imparted imatinib resistance in SupB15WT cells by modulation of growth, survival and migration. When SupB15WT cells were treated with LPA, alone or in combination with imatinib, SupB15WT cell proliferation was increased both in the absence as well as in the presence of imatinib. The dose-dependent increase of proliferation after LPA treatment was 1.9–2.6-fold (1–10μM LPA) in the presence of 1μM imatinib. In addition we performed migration experiments using Transwell assays. We detected a 3-fold increase in migration of SupB15RT vs. SupB15WT cells. We found no influence on apoptosis in imatinib treated SupB15WT cells treated with LPA compared with cells not treated with LPA. Taken together, our results indicate a role of ATX in imatinib-resistant SupB15RT cells, preferentially by stimulating proliferation and migration through LPA signaling via LPA receptors and activation of PI3K and Akt.</jats:p

The Histone Deacetylase Inhibitor LAQ824 Maintains Normal Hematopoietic Progenitor cells (HPC) Associated with Induction of Notch Target Genes and Does Not Eliminate Leukemic HPC in Vitro.

Abstract Introduction: Histone deacetylase inhibitors (DACi) have shown promising antileukemic activity by overcoming the differentiation block and inducing apoptosis in AML blasts. Recent data demonstrating enhanced maintenance and functional capacity of normal, but also leukemic hematopoietic progenitor cells (HPC) by the selective class I DACi valproic acid (VPA) have raised concerns about VPA in AML therapy. As more potent pan-DACi have entered clinical trials, we analysed the impact of the hydroxamic acid LAQ824 on phenotype and function of normal and leukemic CD34+ HPC and studied LAQ824- induced gene expression in the most primitive CD34+CD38- population of normal HPC. Methods: Differentiation and proliferation of CD34+ cells of bone marrow of healthy donors and peripheral blood samples of newly diagnosed AML patients were evaluated after one week of culture in presence of SCF, FLT3 ligand, TPO, IL-3 +/− LAQ824. The effect of LAQ824 on gene expression profiles in normal CD34+CD38− cells was assessed in three independent cell samples following incubation with cytokines +/− LAQ824 for 48 hours using Affymetrix GeneChip Human Genome U133 Plus 2.0 and Gene Spring Software. Serial replating of murine Sca1+Lin- HPC was performed in the presence of SCF, G-CSF, GM-CSF, IL-3, IL-6 +/− LAQ824. Results: Treatment of murine Sca1+Lin- HPC with LAQ824 (10 nM) significantly augmented colony numbers (p&amp;lt;0.01; n=3), and supported colony growth after four cycles of replating whereas no colonies developed in its absence beyond the second plating indicating preservation of functionally active multipotent progenitor cells. LAQ824 (10–20 nM) mediated acetylation of histone H3 in human normal and leukemic HPC. In normal HPC, LAQ824 (0–20 nM) lead to a dose-dependent increase in the proportion of CD34+ cells (20% w/o LAQ824 vs. 36% with LAQ824 20nM, p=0.07) and a significant reduction of CD14+ monocytes (18% vs. 3%, p= 0.02; n=3). The total number of CD34+ cells remained stable up to 10 nM and decreased at 20 nM. Gene expression analysis showed, that LAQ824 (20 nM) lead to an at least 3-fold up-regulation of 221 genes in all three HPC samples tested including HDAC11 and the cell cycle inhibitor p21waf1/cip1 known to be induced by most DACi in HPC. We identified several members of the notch pathway such as mastermind-like protein 2 (MAML2, a component of the active notch transcriptional complex) and notch target genes including the transcription factors HES1, HEY1 and HOXA10 and confirmed increase of protein levels by Western blotting. Reduced gene expression of mini-chromosome-maintenance (MCM) protein family members was observed which - in addition to up-regulation of p21 - has previously been associated with notch-mediated cell cycle arrest. To compare the effect of LAQ824 (20 nM) with VPA (150 ng/ml) on leukemic HPC, cells were cultured for one week with or w/o DACi. Of note, LAQ824 resulted in a 0.8-fold reduction of CD34+ leukemic HPC, while VPA expanded this population 2.2-fold compared with cytokine-treated controls (p=0.03; n=12). CFU numbers growing from CD34+ leukemic HPC in presence of LAQ824 did not differ significantly from controls (n=9). Conclusion: LAQ824 seems to diminish, but not eliminate normal as well as leukemic HPC as determined by phenotypic and functional in vitro analyses. Our gene expression analysis suggested an association with coactivator and target genes of the notch pathway and cell cycle arrest-inducing genes. In contrast to VPA, LAQ824 does not seem to support growth of leukemic HPC which may contribute to its more potent antileukemic effect.</jats:p

CD19-CAR engineered NK-92 cells are sufficient to overcome NK cell resistance in B-cell malignancies

Many B-cell acute and chronic leukaemias tend to be resistant to killing by natural killer (NK) cells. The introduction of chimeric antigen receptors (CAR) into T cells or NK cells could potentially overcome this resistance. Here, we extend our previous observations on the resistance of malignant lymphoblasts to NK-92 cells, a continuously growing NK cell line, showing that anti-CD19-CAR (αCD19-CAR) engineered NK-92 cells can regain significant cytotoxicity against CD19 positive leukaemic cell lines and primary leukaemia cells that are resistant to cytolytic activity of parental NK-92 cells. The ‘first generation’ CAR was generated from a scFv (CD19) antibody fragment, coupled to a flexible hinge region, the CD3ζ chain and a Myc-tag and cloned into a retrovirus backbone. No difference in cytotoxic activity of NK-92 and transduced αCD19-CAR NK-92 cells towards CD19 negative targets was found. However, αCD19-CAR NK-92 cells specifically and efficiently lysed CD19 expressing B-precursor leukaemia cell lines as well as lymphoblasts from leukaemia patients. Since NK-92 cells can be easily expanded to clinical grade numbers under current Good Manufactoring Practice (cGMP) conditions and its safety has been documented in several phase I clinical studies, treatment with CAR modified NK-92 should be considered a treatment option for patients with lymphoid malignancies

Use of a novel histone deacetylase inhibitor to induce apoptosis in cell lines of acute lymphoblastic leukemia.

Background and objectives: Chromatin structure and thereby transcription is controlled by the level of acetylation of histones, which is determined by the balance between histone acetyl transferase (HAT) activity and histone deacetylase (HDAC) activity. HDAC inhibitors are a class of compounds able to regulate gene expression by modulating chromatin structure. There are two major classes of HDAC inhibitors: the hydroxamic acid derivatives such as trichostatin A (TSA) or SAHA, and the butyrates such as phenyl-butyrate. HDAC inhibitors interfere with differentiation, proliferation and apoptosis in tumor cells. Here, we investigated the activity of a new hydroxamic acid derivative, LAQ824, on lymphoblastic cells. Design and methods: Four different pre-B lymphoblastic cell lines: Sup-B15 and TMD-5, both t(9;22) positive, SEM, t(4;11) positive, and NALM-6 cells were exposed to the hydroxamic acid derivatives, LAQ824 and TSA. Histone hyperacetylation, apoptosis, cell cycle and related pathways were assessed by flow cytometry and Western blotting. Results: LAQ824 significantly inhibited the proliferation of leukemic lymphoblastic cell lines. The effect of LAQ824 was due to increased apoptosis accompanied by activation of caspase-3 and caspase-9, cleavage of poly(ADP-ribose)-polymerase (PARP) as well as by down-regulation of Bcl-2 and disruption of the mitochondrial membrane potential. Surprisingly, LAQ824-induced apoptosis was at least partially independent of caspase activation as indicated by the fact that LAQ824-induced apoptosis was inhibited only partially in both t(9;22) positive Sup-B15 and TMD-5 cells, whereas no inhibition was observed in t(4;11) positive SEM cells upon exposure to the polycaspase inhibitor zVAD-fmk. Interpretation and conclusions: Our study establishes that LAQ824 is a promising agent for the therapy of acute lymphoblastic leukemia

Car-engineered nk cells for the treatment of glioblastoma: Turning innate effectors into precision tools for cancer immunotherapy

Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults and currently incurable. Despite multimodal treatment regimens, median survival in unselected patient cohorts is <1 year, and recurrence remains almost inevitable. Escape from immune surveillance is thought to contribute to the development and progression of GB. While GB tumors are frequently infiltrated by natural killer (NK) cells, these are actively suppressed by the GB cells and the GB tumor microenvironment. Nevertheless, ex vivo activation with cytokines can restore cytolytic activity of NK cells against GB, indicating that NK cells have potential for adoptive immunotherapy of GB if potent cytotoxicity can be maintained in vivo. NK cells contribute to cancer immune surveillance not only by their direct natural cytotoxicity which is triggered rapidly upon stimulation through germline-encoded cell surface receptors, but also by modulating T-cell mediated antitumor immune responses through maintaining the quality of dendritic cells and enhancing the presentation of tumor antigens. Furthermore, similar to T cells, specific recognition and elimination of cancer cells by NK cells can be markedly enhanced through expression of chimeric antigen receptors (CARs), which provides an opportunity to generate NK-cell therapeutics of defined specificity for cancer immunotherapy. Here, we discuss effects of the GB tumor microenvironment on NK-cell functionality, summarize early treatment attempts with ex vivo activated NK cells, and describe relevant CAR target antigens validated with CAR-T cells. We then outline preclinical approaches that employ CAR-NK cells for GB immunotherapy, and give an overview on the ongoing clinical development of ErbB2 (HER2)-specific CAR-NK cells currently applied in a phase I clinical trial in glioblastoma patients