Fig 2: Higher Cic expression in Purkinje neurons from the anterior cerebellum of SCA1 mice is associated with the repression of key ion channel genes. (A) Quantification of relative expression of Cic mRNA in the anterior cerebellum and nodular zone of ATXN1[82Q] and wild-type cerebella at P35. (B) Quantification of relative expression of Cic protein in the somata of ATXN1[82Q] and wild-type Purkinje neurons at P35, normalized to values from wild-type anterior cerebellum. (C) Representative confocal images taken from ATXN1[82Q] and wild-type Purkinje neurons at P35 after immunostaining for Cic (green) and calbindin (red, to mark Purkinje neurons). (D) Quantification of relative expression of ATXN1 protein in the somata of ATXN1[82Q] and wild-type Purkinje neurons at P35. (E) Representative confocal images taken from ATXN1[82Q] mice at P35 after immunostaining for ATXN1 (green) and calbindin (red, to mark Purkinje neurons). (F and G) qChIP demonstrating the association of Cic and Atxn1 at the promoter of SCA1-associated genes from sonicated chromatin derived from P14 whole cerebellar extracts. Binding, represented as % input (y-axis) demonstrated for Atxn1 (F) and Cic (G) comparing their relative binding on SCA1-associated genes in Atxn1154Q/2Q mice and wild-type controls and binding over background relative to their respective isotype control IgG (rabbit IgG). (H) Log2 transformation of fold change expression demonstrating the role for the ATXN1-Cic complex in the dysregulation of Cacna1g, Itpr1, Kcnma1 and Trpc3. Each column represents a distinct comparison (numbered by column): 1. ATXN1[82Q] relative to wild-type; 2. ATXN1[82Q]V591A;S602D relative to wild-type and 3. ATXN1[82Q]V591A;S602D relative to ATXN1[82Q]. (I) qRT-PCR for Kcnma1, Cacna1g, Itpr1 and Trpc3 from ATXN1[82Q]V591A;S602D and ATXN1[82Q] cerebella. *Denotes P < 0.05; **denotes P < 0.01; ***denotes P < 0.001; ns denotes P > 0.05; two-tailed Student’s t-test (A and C); two tailed-Student’s t-test with Holm-Sidak correction for multiple comparisons (D–I).

Fig 3: IC overexpression and cell proliferation. a RasB8, U87-EGFRvIII or b U87 were either serum-starved or maintained in 10% FBS, lysed and immunoblotted with indicated antibodies (top panel) or BrdU incorporation assay conducted (bottom panel). c Representative immunohistochemistry images using anti-Ki67 (scale bar, 500 µm) or anti-CIC (scale bar, 1 mm) antibody of high-grade tumors from RasB8 transgenic mice. d RasB8 transfected with or without increasing HA-CIC plasmid lysed and immunoblotted with indicated antibodies. e Quantitative real-time PCR analysis of RasB8 transfected with or without HA-CIC. The graph depicts fold changes in ETV1 expression relative to control. RasB8 transfected with or without HA-CIC plated equally plated and cell viability was assessed at indicated time points (in hours) by trypan blue exclusion or f alamar blue assay (g). h U87-EGFRvIII transfected with or without increasing concentrations of HA-CIC lysed and protein immunoblotted with indicated antibodies. i Quantitative real-time PCR analysis of U87-EGFRvIII transfected with or without HA-CIC. The graph depicts fold changes in ETV1 or -5 expression relative to control. U87-EGFRvIII transfected with or without HA-CIC plated equally and cell viability assessed at indicated time points (in hours) by trypan blue exclusion or j alamar blue assay k. l U87-EGFRvIII transfected with GFP-CIC labeled with eFluor 670 proliferation dye analyzed by flow cytometry. Graphs depict percentage of proliferating GFP-positive versus GFP-negative cells within the same population. Data are representative of at least three independent experiments. U87-Flag-CIC or control cells lysed and m protein lysates immunoblotted with indicated antibodies, n quantitative real-time PCR analysis conducted assessing ETV- or -5 expression or o BrdU incorporation assay. GL261-Flag-CIC or controls were lysed and p immunoblotted with indicated antibodies or q BrdU incorporation assay conducted. Anchorage-independent growth assay of GL261 (r) or U87 (s) cells expressing Flag-CIC showing fold changes relative to control (bottom panel) or phase contrast microscopy images (top panel). All graphs represent mean ± s.e.m. of three independent experiments performed either in octuplet for viability assays or in triplicate for real-time analysis. *P < 0.05 Student’s t-test compared with control. The immunoblot data are representative of at least three separate experiments

Fig 4: A gene signature indicative of Cic LOF is enriched in mouse and human T-ALL driven by inactivation of Cic or activation of the Ras/MAPK pathway. (A) Heat map generated by unsupervised clustering of differentially expressed genes present in a 32-gene Cic LOF signature (CIC_LOF_4), as estimated by RNA-seq from wild-type thymuses and T-ALLs developed in Tmx-treated Ciclox/lox;hUBC-CreERT2+/T as well as KHRasV12;hUBC-CreERT2+/T mice or p53-/- animals at a humane end point. Gene symbols are indicated, and relative expression (log2 fold change) is scaled in color (as indicated) from dark blue (-3) to dark red (+3). (B) Indication of point mutations and gene fusions present in a published data set of human T-ALL samples (from Atak et al. 2013). Samples carrying protein-altering mutations and fusions that are predicted to activate the RAS/MAPK/CIC axis are indicated in dark blue, and those harboring mutations affecting other pathways are indicated in light blue. The mutations affecting specific genes in each sample are indicated in red (only for the RAS/MAPK/CIC group). NUP214-ABL1 and SSBP2-FER represent gene fusions identified in selected samples. Mutations in NOTCH1 are indicated in green. The SUPT1 cell line harbors a gene fusion that causes overexpression of NOTCH1 (light green). (C) Enrichment plot showing significant enrichment (FDR < 25%) of a 143-gene signature indicative of CIC LOF (CIC_LOF_3; left) or a TLX1+ (HOX11+; right) gene signature in human T-ALL samples predicted to have an active RAS/MAPK/CIC axis (from B). (NES) Normalized enrichment score.

Fig 5: Cic inactivation in adult mice causes T-ALL. (A) Tumor-free survival of Cic+/+;hUBC-CreERT2+/T (black dots; n = 13) or Ciclox/lox;hUBC-CreERT2+/T (white dots; n = 11) mice subjected to a continuous Tmx diet at 4 wk of age. (B) Representative image of the thoracic cavity of a Ciclox/lox;hUBC-CreERT2+/T mouse treated for 8 mo with a Tmx diet and sacrificed at a humane end point. (C) H&E as well as CD3 or TDT IHC staining of thymus sections obtained from Cic+/+;hUBC-CreERT2+/T mice at 6 mo of age or Ciclox/lox;hUBC-CreERT2+/T mice at a humane end point. Bar, 50 µm. (D) Representative flow cytometry analyses of CD4+ and CD8+ cells in thymuses obtained from Cic+/+;hUBC-CreERT2+/T mice at 6 mo of age or Ciclox/lox;hUBC-CreERT2+/T mice at a humane end point. (E) Abundance of TCR clonotypes in three independent T-ALL tumors obtained from Tmx-treated Ciclox/lox;hUBC-CreERT2+/T mice (tumors 1–3) or a representative thymus from a Cic+/+;hUBC-CreERT2+/T mouse (wild type). The relative abundance of each clonotype is shown, calculated as the abundance of each clonotype relative to the total abundance of all clonotypes of the same TCR chain in a sample. Clonotypes are plotted in lexicographical order. The read abundance of each clonotype is represented by the size of the symbol (large symbols, 1:1,000,000; small symbols, 1:10,000,000). Clonotypes determined to be dominant are shown in orange. (Circles) a TCR chains; (triangles) ß TCR chains; (cross) ? TCR chains; (X) ambiguous TCR chains.