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Evaluation of WNT pathway transcriptomes in T-cell Acute Lymphoblastic Leukemia by targeted RNA-Seq

ABSTRACT: Wnt/Fzd signaling has been implicated in hematopoietic stem cell maintenance and in acute leukemia establishment. We previously described a recurrent rearrangement involving the WNT10B locus (WNT10BR), characterized by expression of the WNT10BIVS1 transcript variant, in acute myeloid leukemia. To determine the occurrence of WNT10BR in T-cell acute lymphoblastic leukemia (T-ALL), we retrospectively analyzed an italian cohort of patients (n=20) and detected the WNT10BIVS1 with a high prevalence (14/20). To address genes involved in WNT10B molecular response, we designed a Wnt targeted RNA sequencing panel. We identify Wnt agonists and antagonists, from which the expression of FZD6, PLCB4, and PROM1 stand out in WNT10BIVS1-positive patients compared to negative ones. Using MOLT4 and MUTZ-2 as cell models, characterized by the expression of WNT10BIVS1, we found that WNT10B drives major Wnt activation through receipt of ligand to the FZD6 receptor complex. The WNT10B/FZD6 interaction were interfered with by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated inhibition of WNTs secretion. We found that WNT10BIVS1 knockdown, or pharmacological interference by the LGK974 porcupine (PORCN) inhibitor, reduces WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. These results describe the molecular circuit induced by WNT10B and suggest WNT10B/FZD6 as new targets in the T-ALL treatment strategy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE159797 | GEO | 2020/10/22

REPOSITORIES: GEO

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Project description:Nails protect the soft tissue of the tips of digits in humans. In birds and mammals the equivalent claws are used for purposes including capturing prey, digging, climbing, fighting and maintaining dexterity and balance. The molecular mechanism of nail (and claw) development is largely unknown, but we have recently identified a Wnt receptor gene, Frizzled6 (Fzd6), as mutated in a human autosomal-recessive nail dysplasia. To investigate the action of Fzd6 in claw development at the molecular level, we compared gene expression profiles of digit tips of wild-type and Fzd6-/- mice, and show that Fzd6 regulates the transcription of a striking number of epidermal differentiation-related genes. Sixty-three genes encoding keratins, keratin associated proteins, and transglutaminases and their substrates were significantly down-regulated in the knockout mice. Among them, four hard keratins, Krt86, Krt81, Krt34 and Krt31; two epithelial keratins, Krt6a and Krt6b; and transglutaminase1 were known to be expressed in nails and involved in nail abnormality when dysregulated. Immunohistochemical studies revealed decreased expression of Krt86, Krt6b and involucrin in the epidermal portion of the claw field in the knockout embryos. We further show that Dkk4, a Wnt antagonist, was significantly down-regulated in Fzd6-/- mice along with Wnt, Bmp and Hh family genes; and Dkk4 transgenic mice showed a subtly but appreciably modified claw phenotype. Thus, Fzd6-mediated Wnt signaling likely regulates the overall differentiation process of nail/claw formation. Heterozygous Fzd6+/- mice (kindly provided by Dr. J. Nathans) were crossed to generate Fzd6+/-, Fzd6-/- and wild-type offspring. Timed matings were set up to harvest embryos at E14.5, E16.5 and E18.5. The morning after mating was designated as E0.5. Tails and the digital tips from forelimbs of three wild-type and Fzd6-/- mice from each embryonic time-point were excised, immediately frozen on dry ice, and stored at 80 C freezer until use. Genotyping was done as described previously (15). RNA was extracted using Trizol Reagent (Life technologies). RNA from dissected forelimbs from each embryo of the three time-points was used for microarray analysis.

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Evaluation of WNT pathway transcriptomes in T-cell Acute Lymphoblastic Leukemia by targeted RNA-Seq

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