Fig. 2. Acquired resistance is leukemia cell-intrinsic and stable.

Resistance phenotype is preserved upon re-transplantation A Cells isolated at disease stages untreated (grey), sensitive (yellow), persisting (orange), and resistant (red) were re-transplanted into secondary recipient mice. Tumors were allowed to grow for 30–40 days in vivo before treatment was initiated at the same dose, route, and schedule as in the previous passage for 2–3 weeks. Leukemic growth and treatment response was monitored by repetitive imaging. B ALL-199S (n = 6) from Fig. S2B that were previously treated with combination chemotherapy (0.5 mg/kg VCR; 100 mg/kg Cyclo) for 2 weeks and untreated ALL-199 (n = 5) were used. Data is also shown in the middle panel of S2B. C ALL-265P (n = 10) from Fig. S1I that were previously treated with combination chemotherapy (0.3 mg/kg VCR; 70 mg/kg Cyclo) for at least 7 weeks were used. Untreated ALL-265U (n = 16) shown in Fig. S1I were used as control (depicted in grey). D ALL-199R (n = 8) from Fig. 1A–F previously treated with combination chemotherapy for up to 18 weeks (0.15 mg/kg VCR, 70 mg/kg Cyclo) and ALL-199U untreated controls (n = 6) were used. B-D: Upper panels: doubling time was calculated based on imaging values. Box indicates median, 25th, and 75th percentile; whiskers indicate min/max; each dot represents one mouse. **p < 0.01 by unpaired t-test. ns: not significant. Lower panels: treatment response was monitored by repetitive imaging; each dot represents one measurement and each line represents one mouse except for ALL-265U in C, where mean+/− SD is shown. E Resistance remains stable after drug holiday. Resistant derivatives D1-D8 were each transplanted into one mouse, grown to high tumor burden, and each re-transplanted into one next recipient mouse for 3 passages, resulting in a total of 6 months in the absence of treatment. At 4th passage and upon advanced tumor load, mice were treated with combination chemotherapy (0.15 mg/kg VCR, 70 mg/kg Cyclo) used in Fig. 1A–F for 3–8 weeks; data are depicted as in Fig. 1C; each dot represents one measurement and each line represents one mouse. F Partial resistance upon high-dose treatment. Resistant ALL-199 derivatives D1-D8 (n = 1 each) and untreated control cells (ALL-199U; n = 5) were each transplanted into mice; upon advanced tumor load, mice were treated with high-dose combination chemotherapy (0.6 mg/kg VCR, 100 mg/kg Cyclo) for 5 weeks and treatment response was monitored by repetitive imaging; mice engrafted with previously untreated cells were monitored for 5 more weeks to assess putative leukemia re-growth; data are depicted as in Fig. 1C, each dot represents one measurement and each line represents one mouse.