Blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome–positive (Ph1-positive) acute lymphocytic leukemia (ALL) are 2 fatal BCR/ABL-driven leukemias against which Abl kinase inhibitors fail to induce a long-term response. We recently reported that functional loss of protein phosphatase 2A (PP2A) activity is important for CML blastic transformation. We assessed the therapeutic potential of the PP2A activator FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), an immunomodulator in Phase III trials for patients with multiple sclerosis or undergoing organ transplantation, in CML-BC and Ph1 ALL patient cells and in in vitro and in vivo models of these BCR/ABL⁺ leukemias. Our data indicate that FTY720 induces apoptosis and impairs clonogenicity of imatinib/dasatinib-sensitive and -resistant p210/p190^BCR/ABL myeloid and lymphoid cell lines and CML-BC^CD34+ and Ph1 ALL^CD34+/CD19+ progenitors but not of normal CD34⁺ and CD34⁺/CD19⁺ bone marrow cells. Furthermore, pharmacologic doses of FTY720 remarkably suppress in vivo p210/p190^BCR/ABL-driven [including p210/p190^BCR/ABL (T315I)] leukemogenesis without exerting any toxicity. Altogether, these results highlight the therapeutic relevance of rescuing PP2A tumor suppressor activity in Ph1 leukemias and strongly support the introduction of the PP2A activator FTY720 in the treatment of CML-BC and Ph1 ALL patients.

Rapid input-restricted change in gene expression is an important aspect of synaptic plasticity requiring complex mechanisms of post-transcriptional mRNA trafficking and regulation. Small non-coding miRNA are uniquely poised to support these functions by providing a nucleic-acid-based specificity component for universal-sequence-dependent RNA binding complexes. We investigated the subcellular distribution of these molecules in resting and potassium chloride depolarized human neuroblasts, and found both selective enrichment and depletion in neurites. Depolarization was associated with a neurite-restricted decrease in miRNA expression; a subset of these molecules was recovered from the depolarization medium in nuclease resistant extracellular exosomes. These vesicles were enriched with primate specific miRNA and the synaptic-plasticity-associated protein MAP1b. These findings further support a role for miRNA as neural plasticity regulators, as they are compartmentalized in neurons and undergo activity-associated redistribution or release into the extracellular matrix.

Lightning Fast is a sensitive fluorescence‐based stain for detecting proteins in one‐dimensional and two‐dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near‐ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon‐arc lamps; 488 nm and 457 nm Argon‐ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid‐state lasers; 543 nm helium‐neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one‐dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix‐assisted laser desorption/ionization‐mass spectrometry (MALDI‐MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G‐250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI‐MS and Edman‐based sequencing chemistry.

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis, and some acute myeloid leukemias (AML). Although juxtamembrane mutations commonly detected in gastrointestinal stromal tumor are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study, we show that myeloid cells expressing activated c-KIT mutants that are imatinib sensitive (V560G) or imatinib resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with the reduced expression of PP2A structural (A) and regulatory subunits (B55α, B56α, B56γ, and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacologic activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential, and induced apoptosis of mutant c-KIT⁺ cells, while having no effect on wild-type c-KIT cells or empty vector controls. FTY720 treatment caused the dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5, and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT–mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT⁺ cancers. Cancer Res; 70(13); 5438–47. ©2010 AACR.