Figure 2. Acquired resistance is leukemia cell intrinsic and stable.

A – D 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 Figure 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 Figure 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 Figure 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, 25^th and 75^th 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 buden and each retransplanted into one next recipient mouse for 3 passages, resulting in a total of 6 months in the absence of treatment. At 4^th passage and upon advanced tumor load, mice were treated with combination chemotherapy (0.15 mg/kg VCR, 70 mg/kg Cyclo) used in Figure 1A-F for 3-8 weeks; data are depicted as in Figure 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 Figure 1C, each dot represents one measurement and each line represents one mouse.