Fig 1: NIPA deficiency impairs NPM-ALK-mediated colony formation and viability in vitro. (A) Colony formation assay in soft agar of Nipako/ko MEFs retrovirally infected with pBABE-puroRNipa or pBABE-puroRempty vector and MigNPM-ALK or Migempty vector. Seeding of 100,000 cells per well, representative wells shown 18 days after plating. Colonies (Nipako/ko pBABE-puroRNipa MigNPM-ALK) = 58.81 ± 6.13. Colonies (Nipako/ko pBABE-puroRempty MigNPM-ALK) = 28.87 ± 1.67. Results from three independent experiments performed in duplicates. Mig = MSCV-IRES-EGFP. (B) Immunoblot of Nipako/ko MEFs retrovirally infected with pBABE-puroRNipa or pBABE-puroRempty vector, and MigNPM-ALK or Migempty vector ensured correct protein expression. (C) Immunoblot of Ba/F3-cells retrovirally infected with MigNPM-ALK and pLMPmiRmNIPA or pLMPmiRctrl, and of Karpas299-cells retrovirally infected with pLMPmiRhNIPA or pLMPmiRctrl. (D) MTS assay of Ba/F3-cells retrovirally infected with MigNPM-ALK and pLMPmiRmNIPA or pLMPmiRctrl after 24 hours of incubation. Representative experiment shown, results reproduced in four independent experiments performed in triplicates. (E) MTS assay of Karpas299-cells retrovirally infected with pLMPmiRhNIPA or pLMPmiRctrl after 24, 48, and 72 h of incubation at standard conditions. Representative experiment shown, results reproduced in eight independent experiments performed in triplicates. (F) MTS assay of Karpas299-cells retrovirally infected with pLMPmiRhNIPA or pLMPmiRctrl, treated with 0.5 nM of ALK-inhibitor TAE-684, after 24, 48, and 72 h of incubation at standard conditions. Representative experiment shown, results reproduced in three independent experiments performed in triplicates. OD, optical density at 490 nm. *p < 0.05, **p < 0.01, ***p < 0.001. Data shown as mean +SD.
Fig 2: NIPA is associated with “stem-cell-like” features of T-lymphocytes in ALCL-like lymphomas. (A) Representative flow cytometry gating strategy for Lineage- (CD4-CD8-CD25-CD44-) SCA1+ cKIT+ subpopulation in EGFP+ thymic cells of LckCreTG/wtNipawt/wt and LckCreTG/wtNipaflox/flox MSNAIE transplanted mice. EGFP+ Lineage-DN cells were stained for cKIT and SCA1. (B) Representative flow cytometry gating strategy for CLP subpopulation in EGFP+ thymic cells of LckCreTG/wtNipawt/wt and LckCreTG/wtNipaflox/flox MSNAIE transplanted mice. EGFP+ Lin- cKITlow SCA1low cells were stained for Il7Ra. (C) Proportion of subpopulations determined in (A) and (B) in EGFP+ thymic lymphoma cells of LckCreTG/wtNipawt/wt (n = 8) and LckCreTG/wtNipaflox/flox (n = 12) MSNAIE transplanted mice. *p < 0.05, **p < 0.01, ***p < 0.001. Data shown as mean +SD.
Fig 3: Loss of NIPA prolongs survival in short and long latency NPM-ALK driven murine tumorigenesis. (A) Kaplan–Meier survival curve of mice transplanted with 300,000 Nipako/ko and Nipawt/wt bone marrow cells infected with MigNPM-ALK (3%). Median survival was 28.5 days (Nipako/ko , n = 8) vs. 27 days (Nipawt/wt , n = 8). (B) Kaplan–Meier survival curve of mice transplanted with 200,000 Nipako/ko and Nipawt/wt bone marrow cells infected with MigNPM-ALK (0.4%). Median survival was 118 days (Nipako/ko , n = 12) vs. 84 days (Nipawt/wt , n = 12). (C) Number of spleen colonies in mice transplanted with Nipako/ko and Nipawt/wt BMCs infected with MigNPM-ALK at final stage of disease. Mean amount of colonies per spleen were 10 (Nipako/ko , n = 5) vs. 28 (Nipawt/wt , n = 5). Representative spleens shown. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 4: Deletion of Nipa delays lymphoma progression in an ALCL-like mouse model. (A) Kaplan–Meier survival curve of mice transplanted with LckCreTG/wtNipawt/wt MSNAIE and LckCreTG/wtNipaflox/flox MSNAIE bone marrow. Median survival was 121 days (Nipawt/wt , n = 9) versus 143 days (Nipaflox/flox , n = 11). Data from three independent transplantations was analyzed. (B) Representative images of infiltrated organs from mice transplanted with LckCreTG/wtNipaflox/flox MSNAIE BMCs. LN, lymph node; T, thymus; S, spleen. (C) Gel electrophoresis showing lymphoma genotype of representative LckCreTG/wtNipawt/wt and LckCreTG/wtNipaflox/flox MSNAIE transplanted mice in different lymphatic organs, correlated to EGFP-positivity and expression of T-cell markers. (D) Immunphenotyping of representative thymic lymphoma tissue determined by flow cytometry. (E) Mature T-cell distribution determined by flow cytometry for CD4 and CD8 in EGFP+ thymic cells of LckCreTG/wtNipawt/wt (n = 9) and LckCreTG/wtNipaflox/flox MSNAIE (n = 16) transplanted mice. (F) DN T-cell subpopulations determined by flow cytometry due to CD44 and CD25 expression in EGFP+ thymic cells of LckCreTG/wtNipawt/wt (n = 8) and LckCreTG/wtNipaflox/flox MSNAIE (n = 14) transplanted mice. Representative flow cytometry gating strategy on the right. *p < 0.05, **p < 0.01, ***p < 0.001. Data shown as mean +SD.
Fig 5: Mass spectrometry-based proteomic phosphosite analysis identified specific NPM-ALK induced NIPA phosphosites. (A) Overexpressed FLAG-tagged NIPA in 293T cells was immunoprecipitated and used for proteomic phosphosite analysis: Serine residues S24, S338, S344, S354, S370, S381, S387 and S407 appeared significantly upregulated upon NPM-ALK expression with a ratio >2 in more than 3 independent experiments. (B) Log2-fold change revealed S344 (p = 0.0003) to be highly significantly regulated, S407 (p = 0.0084) significant and S24 (p = 0.0338), S359 (p = 0.0268), S370 (p = 0.018), S381 (p = 0.0159) and T387 (p = 0.0319) as significant residues with induced phosphorylation status upon NPM-ALK expression. (C) Schematic overview of the phosphorylation sites within the human NIPA protein. Red: Serine; green: Threonine; NLS: Nuclear localisation signal; Dbox: Destruction box; C3HC: Zink finger binding motif; F-Box: F-Box domain; C: C-terminus; N: N-terminus; blue: Previously identified amino acids phosphorylated at G2/M; black: Possible amino acids phosphorylated by NPM-ALK identified with proteomic phosphosite analysis (* p < 0.05, ** p < 0.01, and *** p < 0.001).
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