T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that is frequently associated with activating mutations in NOTCH1 and dysregulation of MYC. Here, we performed 2 complementary screens to identify FDA-approved drugs and drug-like small molecules with activity against T-ALL. We developed a zebrafish system to screen small molecules for toxic activity toward MYC-overexpressing thymocytes and used a human T-ALL cell line to screen for small molecules that synergize with Notch inhibitors. We identified the antipsychotic drug perphenazine in both screens due to its ability to induce apoptosis in fish, mouse, and human T-ALL cells. Using ligand-affinity chromatography coupled with mass spectrometry, we identified protein phosphatase 2A (PP2A) as a perphenazine target. T-ALL cell lines treated with perphenazine exhibited rapid dephosphorylation of multiple PP2A substrates and subsequent apoptosis. Moreover, shRNA knockdown of specific PP2A subunits attenuated perphenazine activity, indicating that PP2A mediates the drug's antileukemic activity. Finally, human T-ALLs treated with perphenazine exhibited suppressed cell growth and dephosphorylation of PP2A targets in vitro and in vivo. Our findings provide a mechanistic explanation for the recurring identification of phenothiazines as a class of drugs with anticancer effects. Furthermore, these data suggest that pharmacologic PP2A activation in T-ALL and other cancers driven by hyperphosphorylated PP2A substrates has therapeutic potential.

Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

Despite progress in identifying the cellular composition of hematopoietic stem/progenitor cell (HSPC) niches, little is known about the molecular requirements of HSPC support. To address this issue, we used a panel of six recognized HSPC-supportive stromal lines and less-supportive counterparts originating from embryonic and adult hematopoietic sites. Through comprehensive transcriptomic meta-analyses, we identified 481 mRNAs and 17 microRNAs organized in a modular network implicated in paracrine signaling. Further inclusion of 18 additional cell strains demonstrated that this mRNA subset was predictive of HSPC support. Our gene set contains most known HSPC regulators as well as a number of unexpected ones, such as Pax9 and Ccdc80, as validated by functional studies in zebrafish embryos. In sum, our approach has identified the core molecular network required for HSPC support. These cues, along with a searchable web resource, will inform ongoing efforts to instruct HSPC ex vivo amplification and formation from pluripotent precursors.

17, 2009; doi:10.1152/ajpendo.00538.2009.-Wnt/␤-catenin signaling is critical for a variety of fundamental cellular processes. Here, we investigated the implication of the Wnt/␤-catenin signaling in the in vivo regulation of ␤-cell growth and regeneration in normal and diabetic rats. To this aim, TCF7L2, the distal effector of the canonical Wnt pathway, was knocked down in groups of normal and diabetic rats by the use of specific antisense morpholino-oligonucleotides. In other groups of diabetic rats, the Wnt/␤-catenin pathway was activated by the inhibition of its negative regulator GSK-3␤. GSK-3␤ was inactivated by either LiCl or anti-GSK-3␤ oligonucleotides. The ␤-cell mass was evaluated by morphometry. ␤-cell proliferation was assessed in vivo and in vitro by BrdU incorporation method. In vivo ␤-cell neogenesis was estimated by the evaluation of PDX1-positive ductal cells and GLUT2-positive ductal cells and the number of ␤ cells budding from the ducts. We showed that the in vivo disruption of the canonical Wnt pathway resulted in the alteration of normal and compensatory growth of ␤-cells mainly through the inhibition of ␤-cell proliferation. Conversely, activation of the Wnt pathway through the inhibition of GSK-3␤ had a significant stimulatory effect on ␤-cell regeneration in diabetic rats. In vitro, GSK-3␤ inactivation resulted in the stimulation of ␤-cell proliferation. This was mediated by the stabilization of ␤-catenin and the induction of cyclin D. Taken together, our results demonstrate the involvement of the canonical Wnt signaling in the neonatal regulation of normal and regenerative growth of pancreatic ␤-cells. Moreover, we provide evidence that activation of this pathway by pharmacological maneuvers can efficiently improve ␤-cell regeneration in diabetic rats. These findings might have potential clinical applications in the regenerative therapy of diabetes.diabetes; Wnt/␤-catenin pathway; glycogen synthetase kinase-3␤

Ultra-high-dose-rate FLASH radiation therapy has been shown to minimize side effects of irradiation in various organs while keeping antitumor efficacy. This property, called the FLASH effect, has caused enthusiasm in the radiation oncology community because it opens opportunities for safe dose escalation and improved radiation therapy outcome. Here, we investigated the impact of ultra-high-dose-rate FLASH versus conventional-dose-rate (CONV) total body irradiation (TBI) on humanized models of T-cell acute lymphoblastic leukemia (T-ALL) and normal human hematopoiesis.

Acute erythroleukemia (AML-M6 or AEL) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing three genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (e.g. DNMT3A, TET2 or IDH2), and undefined cases with low mutational burden. We established an erythroid vs. myeloid transcriptomics-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, more than 25% of AEL patients, including in the genetically-undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1 binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicates that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.

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Adrenomedullin (ADM) has been shown to mediate multifunctional responses in cell culture and animal system such as regulation of growth and apoptosis. ADM stimulates the proliferation of osteoblasts in vitro and promotes bone growth in vivo. The ability of ADM to influence osteoblastic cell number through inhibition of apoptosis has not yet been studied. To address this question we have investigated its effect on the apoptosis of serum-deprived osteoblastic cells using mouse MC3T3-E1 cells which express both ADM and ADM receptors. Treatment with ADM significantly blunted apoptosis, evaluated by caspase-3 activity, DNA fragmentation quantification and annexin V-FITC labeling. This effect was abolished by the subtype-1 CGRP receptor antagonist, CGRP(8-37). Both ADM and its specific receptor antagonist, the (22-52) ADM fragment exhibited a similar anti-apoptotic effect. Thus, our data suggest that ADM exerts anti-apoptotic effects through CGRP1 receptors. This was substantiated by a similar protective effect of CGRP on MC3T3-E1 cells apoptosis. Accordingly, neutralization of endogenous ADM by a specific antibody enhanced apoptosis. Finally, the selective inhibitor of MAPK kinase (MEK), PD98059, abolished the apoptosis protective effect of ADM and prevented ADM activation of ERK1/2. These data show that ADM acts as a survival factor in osteoblastic cells via a CGRP1 receptor-MEK-ERK pathway, which provides further understanding on the physiological function of ADM in osteoblasts.