Fig. 8.

TCL1A affects ATM functions and cooperates with ATM deficiency. a Protracted TCL1A overexpression in HH T-cell leukemia cells mediates telomere shortening (flow-FISH; Supplementary Fig. 17b–d for controls and qRT-PCR based validations, mean with SEM; Student's t-test). b H₂DCFDA-based measurements of ROS induction upon γ-irradiation (10 Gy) comparing parental Hut78 T-cell leukemia cells to their derivatives of stable TCL1A transfection. Left: examples of H₂DCFDA fluorescence readout 48 h post irradiation, one representative experiment shown. Right: quantification of mean fluorescence intensity (MFI, n = 3 biological replicates, mean with SEM) relative to Hut78 cells without irradiation (p = 0.017, Student's t-test). c Enforced TCL1A expression in HH cells (doxycycline-inducible iHH) mediates higher peak focus counts of DSB marks (γH2AX, RAD51, and TP53BP1) and their impaired resolution in time-lines after washout from a preceding 1 h of Etoposide exposure. Left: quantified focus counts (mean with SEM; Student's t-test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05); right: representative examples (scale bar = 7.5 µm; controls in Supplementary Fig. 17f–h). d Left: setup of MEFs (Rosa-Cre^ERT2;ATM^fl/wt and Rosa-Cre^ERT2;ATM^fl/KD)⁷⁶ stably transfected to express human (h)TCL1A. ATM loss was induced through Tamoxifen treatment over 3 passages resulting in ATM^−/wt and ATM^−/KD cells. Immunoblots in Supplementary Fig. 18a verify reduced ATM protein and overexpression of TCL1A. The KD-mutations D2880A/N2885K correspond to D2870A/N2875K in human ATM. Right: γ-irradiation reduced cell viability (proliferation) as per MTT assay (48 h) with TCL1A mediating a protective effect in scenarios of genetic ATM disruption (mean with SEM; Student's t-tests). e In vivo model of overexpression of hTCL1A and inducible ATM-abrogation. Left: hematopoetic stem cells (HSCs) of Rosa-Cre^ERT2;ATM^fl/fl mice were retrovirally transduced with hTCL1A or a GFP control vector and transplanted into irradiated syngeneic hosts. Recombination of the ATM locus (fl/fl) was induced by Tamoxifen (TM) injections starting 12 weeks after transplantation (1 mg/day i.p. for 5 consecutive days; further details in Supplementary Fig. 18c, d and Methods section). Middle: Kaplan–Meier curve showing accelerated T-cell lymphoma/leukemia onset and shorter animal survival of the ATM^fl/fl/hTCL1A^tg genotype (log-rank tests, time from transplantation to event). Right: evidence of hTCL1A-protein positive T-cells (flow cytometry) in blood and thymus involved by a CD4⁺/8⁺ T-cell tumor (ATM^fl/fl/hTCL1A^tg). H/E stains (scale bar left = 100 µm; right = 400 µm) of one exemplary thymic T-cell lymphoma (ATM^fl/fl/hTCL1A^tg)