Ikaros

The MINO MCL xenograft model was generated by inoculating subcutaneously 6- to 7-week-old nude mice (Shanghai Ling Chang Experimental Animal Co., Ltd.) with 5 × 10⁶ MINO cells. When tumor volume reached 100 to 200 mm³, animals were randomized into five groups of 8 to10 mice each, and were dosed orally with TG-1701 (25, 50, or 100 mg/kg, orally, twice a day), ibrutinib (100 mg/kg, orally, twice a day), or vehicle for 21 days. The tumor volume (V) was calculated as: V = 1/2 × a × b², where a and b represent the length and width, respectively. In REC-1 and UPN-IbruR xenografts, CB17-SCID mice (Janvier labs) were inoculated subcutaneously with 10⁷ REC-1^GFP+Luc+ cells or UPN-IbruR cells and monitored for tumor growth, bioluminescence signal, and vital parameters as described previously (21 (link)), in compliance with the Animal Ethics Committee of the autonomous University of Barcelona (registry number 38/18). Tumor-bearing mice received either TG-1701 (25 mg/kg, every day) or ibrutinib (25 mg/kg, every day) for 17 days. Tumor samples were snap-frozen in OCT medium (Sakura Tissue Tek) or formalin-fixed and paraffin-embedded prior to immunohistochemical (IHC) staining with primary antibodies against Ikaros (Cell Signaling Technology) and CD20 (Beckman Coulter). Preparations were evaluated using an Olympus microscope and MicroManager software.

HEK and Ramos cell lines were obtained from ATCC,¹² (link) TMD8 was a kind gift of dr. Hernandez-Ilizariturry- it´s origin was authenticated in our laboratory by mutational analysis showing the presence of the specific mutation of CD79B gene (Y196H). The cell lines were grown in IMDM media supplemented with 10% FCS, antibiotics (penicillin, streptomycin) and were mycoplasma negative. Human blood was obtained from volunteers under approval of local ethical committee, samples were processed by Ficoll gradient centrifugation and isolated leukocytes were further used for lentiviral infections. Following fluorochrome labeled antibodies were used: Alexa 647 goat anti-mouse (Jackson Immuno), pacific Blue anti-CD4⁺, PerCP anti-CD8⁺, APC anti-CD69, PE anti-CD19 and PE anti-CD20 (all Exbio), for stimulation and western blotting were used unlabeled antibodies specific for: CD3, CD28 (Miltenyi Biotec), TCRzeta (Santa Cruz), Aiolos, Ikaros, pERK, total ERK (all Cell Signaling).