Figure 1.
DEX treatment extends the survival of recipients of T-ALL 8633 and results in the emergence of resistant subclones with diverse Jdp2 integrations. (A) Overview of T-ALL generation in mice infected with the MOL4070 retrovirus and subsequent transplantation and treatment of primary leukemia cells. (B) Kaplan-Meier survival analysis of T-ALL 8633 treated with vehicle (n = 4), DEX (n = 5) or DEX/GDC-0941 (n = 5) (vehicle vs DEX, P < .005; vehicle vs DEX/GDC-0941, P < .005; DEX vs DEX/GDC-0941, P > .05). (C) Southern blot showing patterns of MOL4070 integrations in genomic DNA extracted from leukemia cells harvested from individual moribund recipients of T-ALL 8633 at relapse, after treatment with vehicle, DEX, or DEX/GDC-0941. The arrow highlights the integration observed across all clones, which likely represents Olfr56 (see supplemental Table 1). (D) Jdp2 mRNA expression in parental/vehicle-treated (18B) leukemia 8633 and leukemias isolated from the 5 recipient mice treated with DEX at relapse (19B-23B). Error bars represent the standard deviation of technical triplicates. 22B is the only DEX-treated 8633 subclone without 1 or more Jdp2 integrations. (E) PCR amplification of unique host/virus DNA junction fragments detected by shotgun cloning in relapsed clones 19B, 21B, and 24B based on the data shown in supplemental Table 1. PCR products of the predicted sizes were only amplified from 19B (left), 21B (middle), and 24B (right) using primer pairs specific for each respective integration, which were sequence verified, as shown in supplementary Figs. 1 and 2. The asterisk in the middle panel indicates a background amplification product in 18B, which does not contain the junction fragment observed in 21B. (F) Schematic of the clonal evolution observed in murine RIM-induced leukemogenesis and preclinical trials. Molecular analysis of T-ALL 8633 revealed the same Olfr56 retoviral integration (red lightening bolt, arrow in Figure 1C and supplemental Table 1) and somatic Kdm6a mutation in all vehicle- and DEX-treated leukemias. After treatment with DEX, which significantly prolonged survival (Figure 1B), relapsed leukemia exhibited clonal evolution on Southern blots (Figure 1C) with distinct Jdp2 integrations (colored lightening bolts) that were likely acquired during in vivo treatment (Figure 1E).

DEX treatment extends the survival of recipients of T-ALL 8633 and results in the emergence of resistant subclones with diverse Jdp2 integrations. (A) Overview of T-ALL generation in mice infected with the MOL4070 retrovirus and subsequent transplantation and treatment of primary leukemia cells. (B) Kaplan-Meier survival analysis of T-ALL 8633 treated with vehicle (n = 4), DEX (n = 5) or DEX/GDC-0941 (n = 5) (vehicle vs DEX, P < .005; vehicle vs DEX/GDC-0941, P < .005; DEX vs DEX/GDC-0941, P > .05). (C) Southern blot showing patterns of MOL4070 integrations in genomic DNA extracted from leukemia cells harvested from individual moribund recipients of T-ALL 8633 at relapse, after treatment with vehicle, DEX, or DEX/GDC-0941. The arrow highlights the integration observed across all clones, which likely represents Olfr56 (see supplemental Table 1). (D) Jdp2 mRNA expression in parental/vehicle-treated (18B) leukemia 8633 and leukemias isolated from the 5 recipient mice treated with DEX at relapse (19B-23B). Error bars represent the standard deviation of technical triplicates. 22B is the only DEX-treated 8633 subclone without 1 or more Jdp2 integrations. (E) PCR amplification of unique host/virus DNA junction fragments detected by shotgun cloning in relapsed clones 19B, 21B, and 24B based on the data shown in supplemental Table 1. PCR products of the predicted sizes were only amplified from 19B (left), 21B (middle), and 24B (right) using primer pairs specific for each respective integration, which were sequence verified, as shown in supplementary Figs. 1 and 2. The asterisk in the middle panel indicates a background amplification product in 18B, which does not contain the junction fragment observed in 21B. (F) Schematic of the clonal evolution observed in murine RIM-induced leukemogenesis and preclinical trials. Molecular analysis of T-ALL 8633 revealed the same Olfr56 retoviral integration (red lightening bolt, arrow in Figure 1C and supplemental Table 1) and somatic Kdm6a mutation in all vehicle- and DEX-treated leukemias. After treatment with DEX, which significantly prolonged survival (Figure 1B), relapsed leukemia exhibited clonal evolution on Southern blots (Figure 1C) with distinct Jdp2 integrations (colored lightening bolts) that were likely acquired during in vivo treatment (Figure 1E).

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