Fig 2: ΔEBNA2 EBV plus Myc lymphomas have enhanced expression of GC B cell proteins.Protein extracts were harvested from tumors infected with ΔEBNA2 EBV alone, or co-infected with ΔEBNA2 EBV and Myc vector, and immunoblot analysis was performed to examine expression of various cellular proteins known to be highly expressed in GC B cells and/or BL tumors as indicated. A. Extracts from Donor 1 derived tumors with or without Myc expression were examined for expression of Myc, the EBV LMP1 protein, cyclin D3, cyclin D2, GCSAM or TCL1 as indicated. EBV negative Akata BL cells serve as a positive control for Myc and GC B cell expressed proteins. The same actin immunoblot will be seen in Figs 5D, 6A and 7A, while the LMP1 and Myc blots will be repeated in Figs 5D and 6A. B. Extracts harvested from Myc expressing tumors derived from Donor 1 or Donor 2 as indicated or Donor 1 derived tumors without Myc were examined by immunoblot for expression of Myc, Myb and BACH2 as indicated. Control extracts include the EBV-negative Akata human BL cell line and a BL5 strain EBV infected LCL. C. Extracts harvested from Myc expressing tumors derived from Donor 1 or Donor 2 as indicated or Donor 1 derived tumors without Myc were examined by immunoblot for expression of TCF3 as indicated. Control extracts include the EBV-negative Akata human BL cell line and a BL5 strain EBV infected LCL. D. Extracts from Donor 1 derived tumors with or without Myc expression were examined for expression of Myc, the EBV LMP1 protein, DNMT3B protein, DNMT1 protein and UHRF1 protein as indicated. The same extracts used in Fig 5A were used for this blot and the Myc, LMP1, and actin blots from Fig 5A are reproduced here to show levels of expression in each condition.

Fig 3: PRT543 inhibits tumorigenic properties of ACC cell lines and organoids. (A) ACC cell lines HACC2A and UFH2 were treated with increasing concentrations of PRT543 and relative cell viability was determined on day 7. (B) ACC cell lines were treated with PRT543 or DMSO for 7 days, trypsinized, counted, plated in triplicate in 6-well plates (1 × 106 cells per well) containing inserts and allowed to attach for 12 h. The inserts were removed and the gap was photographed immediately and after 12 h. (C) Gap area in individual wells was measured using ImageJ and relative gap width percentage was calculated as the gap area at 12 h relative to the gap area at 0 h. (D) ACC cell lines HACC2A and UFH2 were treated with indicated concentrations of PRT543 for 7 days, trypsinized, counted and cultured (1 × 104 cells per well) in triplicate into the transwell chambers. After 24 h the membranes were stained with crystal violet and cells that had migrated through the membrane were photographed. (E) The average number of cells per field that migrated through the membrane is shown as a bar chart. (F) RT-PCR analysis of MYB and MYC gene expression in cells treated with PRT543 for 7 days relative to untreated controls. (G) Lysates were collected at end point from cells treated with increasing concentrations of PRT543 and analyzed by western blot for the expression of indicated proteins. GAPDH was used as loading control. (H) Normalized expression of 24 ACC-related genes in HACC2A and UFH2 cells treated with increasing concentrations of PRT543 displayed as a heatmap. Higher or lower expression of genes is indicated with shades of red or blue cells, respectively. (I) Two ACC organoid models were treated with increasing concentrations of PRT543 and relative cell viability was determined on day 7. (J) RT-PCR analysis of MYB and MYC gene expression in organoid models treated with PRT543 relative to untreated controls. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig 4: Both Myc and Myb are involved in altering expression of cellular genes in the ΔEBNA2 EBV plus Myc lymphomas.A. P493-6 cells were treated with or without doxycycline for 3 days, and then protein extracts were harvested to examine expression of various cellular proteins and EBV proteins as indicated. Mutu I is an EBV+ human BL cell line with type 1 latency. The blots shown on the left and top right used the same set of extracts and have the same actin blot. The numbers below each immunoblot quantify the results using Image Studio Lite software to normalize the levels of STAT3, Src, Myb, IL7R, UHRF1, DNMT3B, p100, and p52 expression to actin expression. Results are presented as the ratio of STAT3, Src, Myb, IL7R, UHRF1, DNMT3B, p100, and p52 expression in doxycycline-treated cells relative to untreated P493-6 cells, after being normalized to actin. Values of untreated cells are set as 1. The same extracts are used in S5 and S7 Figs and the actin blot is reproduced there. B. Immunoblot analysis was performed to compare expression of various different GC B cell proteins as indicated in stable tumor-derived cell lines (N1, N3, N4) versus the P493-6 cell line (with Myc expression turned on). The Mutu I BL line and the BL5 LCL line serve as positive and negative controls, respectively, for GC protein expression. C. EBV positive Akata and Mutu I BL cell lines were infected with lentiviral vectors expressing a non-targeting (“NT”) control shRNA or Myb-targeting shRNAs (left panel) or with a Myb-expressing or control lentivirus vector (right panel). Three days later, cells were treated with puromycin for 2 days prior to harvesting protein extracts for immunoblot analysis. The effect of Myb knockdown or Myb over-expression in the BL cell lines on the levels of various cellular proteins regulated by Myc expression is shown. The numbers below each immunoblot quantify the results using Image Studio Lite software to normalize the levels of Myb, Myc, CD179B, Cyclin D3, UHRF1, Rag1, and DNMT3B expression to actin expression. Results are presented as the ratio of Myb, Myc, CD179B, Cyclin D3, UHRF1, Rag1, and DNMT3B expression in Myb knockdown (or Myb over-expression) conditions relative to control conditions after being normalized to actin. Values of control conditions are set as 1. D. EBV positive Akata and Mutu I BL cell lines were infected with lentiviral vectors expressing a non-targeting (“NT”) control shRNA or Myb-targeting shRNAs and immunoblots performed to examine expression of Myb, STAT3, and Src as indicated. Akata BL line and the BL5 LCL line serve as positive and negative controls for protein expression. The same actin immunoblot will be seen in Fig 9F.

Fig 5: Activated STAT3 and Src regulate LMP1 expression in BL cell lines.A. Protein extracts were isolated from stable cell lines derived from Donor 2 ΔEBNA2 EBV plus Myc tumors (grown off the feeder layer) and immunoblot analysis was performed to examine expression of Myc, LMP1, total STAT3, phosphorylated STAT3, and total Src. The EBV negative Akata BL line and a BL5 EBV infected LCL serve as controls. B. Mutu I BL cells were transfected with a vector control, a STAT3 expression vector alone, a Src (constitutively active) expression vector alone, or both STAT3 and Src expression vectors. Two days later protein extracts were harvested and immunoblot analysis performed to examine expression of total STAT3, phosphorylated STAT3, Src, and LMP1 as indicated. C. Mutu I BL cells were infected with lentiviral vectors expressing a non-targeting control shRNA (NT) or STAT3 targeting shRNAs (left panel) or NF-κB2 (p100/52) targeted shRNAs (right panel). Three days later cells were treated with puromycin for 2 days, and then grown on or off an IL40/IL21 expressing feeder cell layer for another 2 days before harvesting protein extracts for immunoblot analysis. The levels of STAT3, LMP1, EBNA2, and p100/52 expression are shown for each condition as indicated. The numbers below each immunoblot in the left panel quantify the results using Image Studio Lite software to normalize the levels of STAT3 and LMP1 expression to actin expression. Results are presented as the ratio of STAT3 and LMP1 expression in STAT3 knockdown conditions relative to control (NT) shRNA, after being normalized to actin. Values of control conditions are set as 1. D. EBV+ Akata BL cells were infected with a lentivirus vector expressing LMP1 or a control lentivirus and two days later cells were treated with puromycin for 3 days. Protein extracts were harvested from puromycin resistant cells and immunoblot analysis performed to examine the levels of LMP1, total STAT3, phosphorylated STAT3, Src, and Myc as indicated. E. Cell lines derived from ΔEBNA2 EBV plus Myc lymphomas or Mutu I BL cells were treated with or without 5’azacitidine for 3 days and then protein extracts harvested for immunoblot to examine expression of LMP1, BZLF1, EBNA2, total STAT3, phosphorylated STAT3 or Src as indicated. F. Extracts isolated from Akata BL cells or Mutu I cells treated with non-targeting shRNA or Myb targeting shRNA (the same extracts used in Fig 8D) were examined by immunoblot analysis for expression of Myb, LMP1, EBNA2 and BZLF1 as indicated. EBV-neg Akata BL cells and a BL5 EBV-infected LCL serve as controls.