Supplemental Information Venetoclax is effective in small cell lung cancers with high BCL-2 expression Timothy L. Lochmann, Konstantinos V. Floros, Mitra Naseri, Krista M. Powell, Wade Cook, Ryan J. March, Giovanna T. Stein, Patricia Greninger, Yuki Kato Maves, Laura R. Saunders, Scott J. Dylla, Carlotta Costa, Sosipatros A. Boikos, Joel D. Leverson, Andrew J. Souers, Geoffrey W. Krystal, Hisashi Harada, Cyril H. Benes, and Anthony C. Faber Supplemental Figure S1. Venetoclax sensitivity and BCL-2 mRNA correlation analysis. (A) LN IC50 waterfall plot of 26 tumor types from Fig. 1A. (B) Correlation analysis of BCL2 mRNA expression from the GDSC compared to venetoclax sensitivity, plotted as AUC vs BCL2 mRNA (left panel) or LN IC50 vs BCL2 mRNA (right panel). Linear regression is plotted as a red line. Correlation was determined using Spearman’s correlation. Supplemental Figure S2. Cell viability assays of border line venetoclax-sensitive and - resistant SCLC cell lines and combination of venetoclax with chemotherapy. 5-day CellTiter-Glo assays were performed on the sensitive (AUC=0.965) NCI-H187 cell line (A), and the resistant (AUC=0.987) NCI-H525 cell line (B). Error bars are plotted as standard error. (C) A dose matrix of Venetoclax and the combination of Cisplatin and Etoposide was performed on the venetoclax-sensitive DMS-53 cell line, venetoclax-resistant, low BCL-2 NCI- H2171 and NCI-H82 cell lines, and the venetoclax-resistant high BCL-2 NCI-H1048, NCI- H1688, and NCI-H211 cell lines (displayed concentrations are µM). Percent of the excess over the Bliss at each dose of the drug is presented. Bliss scores greater than zero, close to zero, and less than zero represent synergy, additivity, and antagonism, respectively. Supplemental Figure S3. Correlation analysis of BCL-2 family members to venetoclax sensitivity in SCLC cell lines. Correlation analysis of PMAIP1 (encodes NOXA, top left), BCL2L1 (encodes BCL-XL, top right), MCL1 (bottom left), or BCL2L11 (encodes BIM, bottom right) mRNA expression from GDSC compared to venetoclax sensitivity, plotted as AUC vs BCL2 mRNA (A) or LN IC50 vs BCL2 mRNA (B). Linear regression is plotted as a red line. Correlation was determined using Spearman’s correlation. Supplemental Figure S4. Comparison of BCL-2 family members to venetoclax sensitivity in SCLC cell lines. (A) Top and bottom quartiles were determined for PMAIP1 (encodes NOXA, top left), BCL2L1 (encodes BCL-XL top right), MCL1 (bottom left), or BCL2L11 (encodes BIM, bottom right). The AUC to venetoclax was plotted for each quartile. (B) Top and bottom quartiles were determined for BCL2 and PMAIP1 (top left), BCL2 and BCL2L1 (top right), BCL2 and MCL1 (bottom left), or BCL2 and BCL2L11 (bottom right). The AUC to venetoclax was plotted for each quartile. The Mann-Whitney U test was used to determine p-values. Supplemental Figure S5. BCL-2 mRNA expression is increased in SCLC compared to other cancer types. (A) Left panel shows BCL2 mRNA expression of SCLC, NSCLC, and other solid tumors from the GDSC RNA expression database. Right panel is a BCL2 mRNA comparison with data from the CCLE RNA expression database. (B and C) Comparison of BCL2 mRNA expression in NSCLC versus SCLC. Determination of p-values was performed using the Mann-Whitney U test. Supplemental Figure S6. Cell viability of venetoclax-sensitive and -resistant SCLC cell lines. 3-day CellTiter-Glo assays were performed on venetoclax-sensitive DMS-53 (A) and NCI- H510A (B) cell lines, -resistant NCI-H82 (C) cell line and an ex vivo cell culture of the LU5263 PDX model (D). Error bars are plotted as standard error. Supplemental Figure S7. Weight profiles of xenograft and PDX models treated with venetoclax. Weight is expressed as a percentage of initial weight in DMS-53 (A), LU5263 (B), LU86 (C), and LU95 (D) mouse models during venetoclax treatment. Supplemental Figure S8. The PDX models from this study express high levels of BCL-2. Western blot of the high BCL-2 expressing PDX model tumors compared to venetoclax-sensitive (NCI-H510A) and -resistant (NCI-H82 and NCI-H2171) cell lines. Expression of the BCL-2 family proteins in SCLC cell lines was determined by Western blot analyses using the indicated antibodies. A B LN IC venetoclax AUC 50 Biliary Tract Bone p=0.000094 r=-0.52 Brain BCL2 Breast Cervix mRNA Esophagus Head & Neck Intestine Kidney Liver SCLC LN IC50 NSCLC p=0.00025 r=-0.49 Misc BCL2 Muscle Nervous System Ovary mRNA Pancreas Pleura Prostate Sup Fig. S1 Sup Fig. Skin Stomach Testes Thyroid Urinary Track Uterus Vulva ANCI-H187 B NCI-H524 150 150 100 100 50 50 % viablecells 0 % viablecells 0 0 M 0 M M nM nM nM n nM µM nM nM nM n 7 .3 6 5 7 4µM3µM 7 .3 6 .5 6 1 3 8. .0 .3 10 .6 1 3 10µ 3. 1 34 1 3 3 1 347nM1.04µM3.33µM venetoclax10 venetoclax108 C DMS-53 NCI-H2171 cisplatin (µM) 1 3 10 cisplatin (µM) 1 3 10 etoposide (µM) 1.5 4.5 15 etoposide (µM) 1.5 4.5 15 0.3 -28.74 -12.12 4.94 0.3 -4.03 -8.73 -0.45 1 -25.64 -23.29 9.00 1 -21.77 -11.76 -1.32 3 -24.74 -32.99 6.94 3 -11.30 -11.35 -2.00 venetoclax (µM) 10 -20.11 -20.66 6.63 venetoclax (µM) 10 -3.20 -3.23 -2.32 NCI-H82 NCI-H1048 cisplatin (µM) 1 3 10 cisplatin (µM) 1 3 10 etoposide (µM) 1.5 4.5 15 etoposide (µM) 1.5 4.5 15 0.3 0.73 -8.10 1.97 0.3 -3.71 -1.96 2.05 1 -3.70 -12.80 -7.10 1 -3.71 -4.08 1.76 3 0.74 0.44 8.93 3 0.21 -3.07 1.06 venetoclax (µM) 10 17.70 1.27 7.37 venetoclax (µM) 10 -16.98 -13.57 -10.45 NCI-H1688 NCI-H211 cisplatin (µM) 1 3 10 cisplatin (µM) 1 3 10 etoposide (µM) 1.5 4.5 15 etoposide (µM) 1.5 4.5 15 0.3 -4.20 -1.56 -3.43 0.3 -6.80 -14.91 5.34 1 -13.49 -8.05 4.98 1 -0.06 -19.98 6.74 3 6.01 11.61 17.77 3 -6.93 3.66 15.13 venetoclax (µM) 10 22.86 13.12 18.18 venetoclax (µM) 10 -19.57 -9.75 8.07 Sup Fig. S2 A n.s. n.s. r=-0.20 r=-0.061 AUC AUC PMAIP1 mRNA BCL2L1 mRNA n.s. n.s. r=0.22 r=-0.25 AUC AUC MCL1 mRNA BCL2L11 mRNA B n.s. n.s. r=-0.12 r=-0.084 50 50 LN IC LN IC PMAIP1 mRNA BCL2L1 mRNA n.s. n.s. r=0.27 r=-0.13 50 50 LN IC LN IC MCL1 mRNA BCL2L11 mRNA Sup Fig. S3 A PMAIP1 mRNA BCL2L1 mRNA n.s. 1.2 n.s. 1.0 AUC 0.8 AUC 0.6 0.4 Bottom Top Bottom Top Quartile Quartile Quartile Quartile MCL1 mRNA BCL2L11 mRNA 1.2 n.s. 1.2 n.s. 1.0 1.0 AUC AUC 0.8 0.8 0.6 0.6 0.4 0.4 Bottom Top Bottom Top Quartile Quartile Quartile Quartile B BCL2*PMAIP1 mRNA BCL2/BCL2L1 mRNA p=0.00038 p=0.0015 1.2 1.2 1.0 1.0 0.8 0.8 AUC AUC 0.6 0.6 0.4 0.4 Bottom Top Bottom Top Quartile Quartile Quartile Quartile BCL2/MCL1 mRNA BCL2*BCL2L11 mRNA n.s. 1.2 1.2 p=0.0019 1.0 1.0 0.8 0.8 AUC AUC 0.6 0.6 0.4 0.4 Bottom Top Bottom Top Quartile Quartile Quartile Quartile Sup Fig. S4 A p=1x10-15 p<1x10-15 p<1x10-15 p=1x10-9 p=0.00013 p=0.045 mRNA mRNA BCL2 BCL2 B C Rohrbeck et al. Bhattacharjee et al. p<0.0001 6 p<0.0001 4 2 mRNA mRNA 0 -2 BCL2 BCL2 -4 -6 Sup Fig. S5 A NCI-H510A % viable cells D C 0 3.67nM B 11.3nM 36nM venetoclax108.5nM 150 347nM 1.04µM 100 NCI-H82 3.33µM 10µM % viable cells 50 DMS-53 % viable cells 0 0 3.67nM 0 11.3nM 3.67nM 36nM 11.3nM venetoclax108.5nM 36nM 347nM venetoclax108.5nM 1.04µM 347nM 3.33µM LU5263 – 1.04µM 10µM 3.33µM 10µM % viable cells ex vivo 0 3.67nM 11.3nM 36nM venetoclax108.5nM 347nM 1.04µM 3.33µM 10µM Sup Fig. S6 A B DMS-53 LU5263 control control venetoclax venetoclax % of initial %weight of initial %weight of initial Days of treatment Days of treatment C D LU86 LU95 control control venetoclax venetoclax % of initial %weight of initial %weight of initial Days of treatment Days of treatment Sup Fig. S7 MCL-1 BCL-XL BCL-2 BIMEL NOXA BAX GAPDH Sup Fig. S8.