Supplemental Material

Tables

Supplemental Table S1: Stromal Dependent PDX samples

Patient Sample / Tissue / Molecular Lesions / Stage
PAUXZX / BM / ETV6/ABL1 / Diagnosis
ICN1 / BM / BCR-ABL p210 / Diagnosis
MXP5 / BM / BCR-ABL p190; PAX5 deletion (Exons 2-6) / Diagnosis
SFO2 / BM / BCR-ABL p210
(no ABL kinase mutations) / Relapse (Dasatinib)

Figures

Supplemental Figure S1: Protection by mTOR inhibition is dose dependent

(A)Viability of BV173 cells treated with 30 nM MTX for 48 hours with increasing mTOR inhibition by allosteric inhibitor rapamycin, TOR-KI MLN0128 and TOR-KI AZD8055. (B) Viability of BV173 cells treated with 100 nM MLN0128 and increasing MTX for 48 hours or 6-MP for 72 hours. (C) BV173 cells treated with methotrexate for 48 hours in presence or absence of 150 nM NVP-BEZ-235. (D) SUP-B15 cells treated with various concentrations of MLN for 48 hours. (E) BV173 and (F) SUP-B15 cells treated with methotrexate for 3 days with or without 100 nM MLN0128. Cells were allowed to regrow without drugs for 7 days and counted. At least 3 replicates, mean±SEM.*p<0.05 and ***p<0.0005.

Supplemental Figure S2: Effect of mTOR inhibition on other chemotherapies

(A) BV173 cells and (B) SUP-B15 cells were treated with various chemotherapeutic agents in combination with mTOR inhibitors 100 nM MLN0128 or 10 nM rapamycin. Vinc, AraC, Etop, and Dex treatments were for 48 hours. DNR treatment was for 24 hours. (C)MLN0128 (100 nM) effect on cytotoxicity of araC was assessed in Ph-like PAUXZX and three Ph+ short-term cultured patient cells. For patient cellson OP9 stroma, 1 M AraC treatment was for 72 hours. All chemotherapy agents were used near their IC50 to easily detect protection or sensitization. Results are from at least 3 replicates for each drug. In all figures unpaired t-tests of “chemo” versus “chemo+mTOR inhibitor” were performed for each drug, mean±SD, *p<0.05, **p<0.005, ***p<0.0005.

Supplemental Figure S3: Titration of PI3K/AKT/mTOR inhibitors

Western blot of SUP-B15 cells treated with (A) GDC-0941, (B) AKTi-VIII, MK-2206, (C) MLN0128 and (D) rapamycin for 6 hours.

Supplemental Figure S4: Dose of dasatinib used inhibits ABL activity in Ph+ lines

(A) BV173 (Ph+) and SUP-15 (Ph+) cells were treated with 3 nM dasatinib for 2 hours. Effect on ABL kinase activity was measured by p-ABL and p-STAT5. (B) Western blot analysis of dasatinib effect on ABL (pSTAT5), mTORC1 (p-S6 and p-4E-BP) and TORC2 (pAKT and pPRAS40) kinase activity in SUP-B15 cells after 6 hours treatment. Viable number of BV173 (C-D) or SUP-B15 (E-F) cells treated with 30 nM MTX or 1 M 6-MP in combination with 100 nM MLN0128 or 3 nM dasatinib counted by Tryphan blue staining. (G) SUP-B15 and (H) BV173 cells treated with 30 nM MTX and increasing amounts of dasatinib for 48 hours. n=3, mean±SD.

Supplemental Figure S5: Killing effect of dasatinib alone can counteract the protection by mTOR inhibition

(A) Western blot of ABL (p-STAT5) and mTORC1 (p-S6) activity after 6 hours of treatment with dasatinib or 100 nM MLN0128 in ICN1 cells. (B) Effect of 100 nM dasatinib on cytotoxicity of 6MP and MTX (treated for 5 days) in ICN1 PDX cells. (C) Western blot of PAUXZX and MXP5 PDX cells after 6 hours of dasatinib and 100 nM MLN0128 treatment. (D) Viability of PAUXZX and MPX5 cells treated with 6MP and MTX for 5 days in presence or absence of 100 nM dasatinib. Unpaired t-tests of “chemo” versus “chemo+dasatinib” were performed for each drug. Results are from at least 3 replicates for each drug, mean±SD, *p<0.05, **p<0.005, ***p<0.0005.

Supplemental Figure S6: Palbociclib alone is not cytotoxic

(A) BV173 and (B) SUP-B15 were treated with Palbociclib for 48 hours followed by Annexin-V/PI staining. n=3, mean±SD.

Supplemental Figure S7: Cell cycle inhibition reduces DNA damage by maintenance drugs

(A) BV173 or (B) SUP-B15 cells were treated with 30 nM MTX for 48 hours with 100 nM MLN0128or3 nM dasatinib. DNA damage was measured by intracellular staining of phospho-histone H2AX. (C) BV173 cells were treated with 10 μM 6-MP for 48 hours in presence of titrated palbociclib or 100 nM MLN0128. (D) Similarly, SUP-B15 cells were treated with MTX for 48 hours. Phospho-CHK1 and phospho-histone H2A.X serve as indicators of DNA damage caused by 6-MP or MTX. DNA damage experiments were all done in the presence of 20 μM QVD-Oph (caspase inhibitor) to rule out effect of activated caspases on DNA damage.

Supplemental Figure S8: mTOR inhibition decreases DHFR expression in some PDX lines

Western blot of patient PDX lines treated with 100 nM MLN0128 for 24 hours. Treatment of BV173 with 30 nM methotrexate serves as positive control for high DHFR expression.

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