SUPPLEMENTARY MATERIALS for Keysar et al.

SUPPLEMENTARY METHODS

Generation of PDX models

A clinical pathologist performed gross dissection of the originating human tumors after resection and

non-diagnostic, non-necrotic portions were utilized. Tumors were placed in collecting medium consisting of

DMEM supplemented with 10% fetal bovine serum (FBS), 200units/mL penicillin, and 200µg/mL

streptomycin, and cut into 3x3x3mm pieces. Up to five 7-10-week-old female Athymic Nude-Foxn1nu mice

(Envigo, Denver, CO) with two tumors each were implanted per patient case in this initial engraftment phase

(1st generation). The right and left hind flanks were sterilized and small incisions were made to create

subcutaneous pockets. Prepared 3x3x3mm tumor pieces were dipped in Matrigel (Corning, Corning, NY) and

inserted into the pocket. When tumors reach 1,500 mm3 they were passed to a second colony of animals (5-10

animals per case; 2nd generation) just as the original human tumors were. Patient cases (4th-8th generation) were

expanded into larger cohorts for CSC isolation or efficacy studies. The authenticity of PDX tumor origin was

confirmed by comparison of 16 short tandem repeat (STR) loci between PDX and patient blood and/or tumor

upon initial engraftment and every 3-4 passages in mice.

Cell growth assay (CellTiter-Glo)

Cell growth assays were performed by seeding the indicated HNSCC cells at 4000 cells/well in 96-well

plates and allowed to grow overnight. Cells were then treated with a dose range of SVC112 (3 to 10,000nM

final) and cells were allowed to grow for 72h. Cell growth was determined with CellTiter-Glo (Promega,

Madison, WI) following manufacture’s protocols and as previously described (1). SVC112 IC50 values were

calculated with Prism software using non-linear regression.

Sulforhodamine B Colorimetric assay (SRB)

Cells (3,000-6,000) were plated in 96-well plates and incubated overnight. Drug was added and plates

were incubated for to 96h. Cells were fixed with 50µl of 10% TCA at 4°C (30min), washed 5X with dH20,

1

70µl/well SRB reagent was added, wells were washed 5X with 1% acetic acid, 200µl/well 10mM Tris base was

added, plates were shaken at 40rpm at RT (15min), and absorbance was measured using a Synergy 2 microplate

reader (Bio-Tek, Winooski, VT). IC50s were calculated by four parameter nonlinear regression, log (inhibitor)

vs. response, using GraphPad Prism v8.0. Calculated values are reported as IC50± 95% confidence interval

(C.I.).

Clonogenic assays

Detroit 562, FaDu, BJ, Detroit 551, IMR-90 and Normal Human Bronchial Epithelial cells (ATCC)

were seeded in 6-well plates and grown overnight. Cells were irradiated (1, 2, 4, 6, 8, and 10Gy), immediately

followed by treatment with a dose-range of SVC112 (5-300nM final). After 24h, drug-containing media was

replaced with fresh media and incubated for 9 days. Cells were fixed with TCA, stained with sulforhodamine B

(SRB) and imaged (Olympus SZX12 Stereo Microscope). SRB was solubilized and quantified in a plate reader

(Synergy 2, BioTek). The ability to SVC112 to enhance the effect of radiation was measured in terms of Dose

Modifying Factor (DMF). Calculation of DMFs is detailed in Radiobiology for the Radiologist (2). DMF

reflects how much more potent radiation is when the drug is present than when it is not present. DMF is

typically measured by performing a radiation dose-response analysis using the clonogenic endpoint, with and

without drug (for example, Fig. 1H). Clonogenic survival is then normalized to the no drug/no radiation value

and plotted in a log-linear graph. DMF is computed as the ratio of the slope of the survival curves with and

without drug. Drugs that enhances the effect of radiation show DMF>1.

For washout clonogenic assays, cell cultures were treated with SVC112 (100 or 1,000nM) for 24h

before washing twice with warm PBS and adding fresh growth media. Cells were collected at 0, 6, 24, and 48h

following drug washout, counted, and seeded in 6-well plates. Cells were fixed in 10% formalin on day 10and

stained with 0.5% crystal violet for 15min for colony counting.

Cell line xenograft efficacy study

2

For cell line xenograft experiments FaDu cells were implanted subcutaneously with treatment beginning

14 days later. Mice were irradiated (2Gy) twice per week. Once weekly Cisplatin (1mg/kg) was administered

I.P. immediately before the first weekly irradiation, while once weekly SVC112 (20mg/kg) was administered

I.P. immediately following the second weekly dose of radiation. All treatments lasted four weeks.

Fluorescence activated cell sorting (FACS)

Tumor tissue was finely minced with a scalpel and dissociated in DMEM containing 1mg/ml

collagenase IV (Worthington, Lakewood, NJ) at 37°C for 1.5h. Cells were filtered (40µm) and red blood cells

were lysed in ACK buffer (Life Technologies, Carlsbad, CA). Staining with Aldefluor (Stem Cell Technologies,

Vancouver, Canada) followed the manufacturer’s instructions. Briefly, cells were suspended in Aldefluor

staining buffer containing Aldefluor reagent (5µl/ml) and incubated at 37oC for 30min. DEAB (N,N-

diethylaminobenzaldehyde) was used as a negative control for setting gates. Following incubating with

Aldefluor, cells were stained with the following antibodies; IgG controls, 1:10 anti-human CD44 (BD

Biosciences, San Jose, CA), as well as 1:100 anti-mouse H-2Kd (BioLegend, San Diego, CA), 1:100 anti-

mouse CD31 (BioLegend) 1:100 anti-mouse CD34 (BioLegend), 1:100 anti-mouse CD45 (BioLegend) for

negative selection of mouse lineage cells, at the dilution of 100µl/1x106 cells. DAPI was use as a viability stain

to negatively select dead cells. Cell sorting was performed using a MoFlo XDP (Beckman Coulter, Fort Collins,

CO) and flow cytometric analysis was completed on a CyAn ADP (Beckman Coulter).

3

SUPPLEMENTARY FIGURES

Supplementary Figure 1. Effects of SVC112 on cancer cell proliferation and response to radiation. (A)

SVC112 depletes Myc and cyclin D1 in HNSCC cells. (B) Clonogenic survival of Det562 HNSCC cells after

treatment with SVC112 and radiation according to schedules shown. Survival was normalized to no drug/no 4

radiation controls. (C) SRB analysis of 049C cells treated with SVC112 and HHT. (D) Cell growth analysis for

cancer (013C, 036C, 049C, 067C) and CAF (013CAF, 036CAF, 067CAF) cell lines seeded at and initial

density of 2x104 cells per well. (E) Plasma concentrations of SVC112 after a single IP injection of 40 mg/kg in

mice. (F) Comparison of HNSCC cancer cell line sphere formation and growth inhibition by SVC112 and HHT.

(G) Decreased sphere formation following SVC112 treatment + 4Gy radiation as compared to radiation alone.

(HI) Relative Myc and Sox2 protein levels in CSCs sorted from PDX tumors compared to the non-CSC tumor

population. (IJ) SVC112 (100nM for 24h) decreases Myc and Sox2 levels in ex vivo cultures of PDX sorted

CSCs. Graphed results are presented as mean ±SD. Statistical significance was calculated by two-tailed

Student’s t test (* P≤ .05, ** P≤ .01). HNSCC=head and neck squamous cell carcinoma, SRB=sulforhodamine

B colorimetric assay, CAF=cancer associated fibroblast. HHT=homoharringtonine, CSC=cancer stem cell,

PDX=patient-derived xenograft.

5

Supplementary Figure 2. Efficacy studies assessing the effects of radiation +/- SVC112 in HNSCC PDX

models. and (A) Regrowth of individual CUHN036, CUHN047, CUHN004, and CUHN013 tumors treated

with vehicle control, SVC112, 4Gy, or 4Gy + SVC112 (≥9 tumors per treatment arm). (B) CUHN036 and

CUHN013 PDX models were treated with radiation and SVC112 alone and in combination. Tumor bearing

animals were irradiated (4Gy x-rays) and treated with SVC112 once weekly. SVC112 (CUHN036 = 80mg/kg,

6

CUHN013 = 60mg/kg) was delivered at the time of irradiation and 6h after irradiation (≥10 tumors per

treatment arm). Treatment vs Control calculations for each tumor at day 28 of the study. Negative values

(dashed red line) represent tumors that regressed with treatment. (C) The effects of SVC112 (20mg/kg once

weekly) when combined with radiation (2Gy twice weekly) + cisplatin (1mg/kg once weekly) was assessed in

FaDu cell line xenografts. (D) Toxicity was assessed by the change in animal body weight over 28-day efficacy

studies for cases CUHN013 and CUHN036 treated weekly and twice weekly (biw), and CUHN004 and

CUHN047 treated twice weekly (≥5 animals per treatment arm). (E) Cleaved caspase-3 increases in radiation

and combination treated CUHN036 and CUHN047 tumors. 400x magnification, scale bars = 100µm. Graphed

results are presented as mean ±SD. Statistical significance was calculated by two-tailed Student’s t test (*

P≤ .05, ** P≤ .01). HNSCC=head and neck squamous cell carcinoma, PDX=patient-derived xenograft.

7

Supplementary Figure 3. Ribo-seq analysis of HNSCC cells treated with SVC112. (A) Enrichment of

Ribosome protected mRNA fragments (RPF, normalized to RNA-seq) for genes following treatment (6h) with

SVC112. FPKM RNA-seq values and IRES prediction for each gene. (B) GSEA pathways of RPFs ugregulated

in cells treated with SVC112. (C) SVC112 treatment suppresses mRNA levels of ALDH1A1 but not 8

proliferation and CSC-related transcription factors. Graphed results are presented as mean ±SD. Statistical

significance was calculated by two-tailed Student’s t test (* P≤ .05, ** P≤ .01). FPKM=fragments per kilobase

of exon per million reads mapped, IRES=internal ribosome entry site.

9

Supplementary Figure 4. Effects of SVC112 on protein synthesis and assessing the stability of SVC112 in

culture. (A) Relative levels of methionine analog incorporation (Click-iT assay, relative fluorescence)

following a 1.5h incubation period for HNSCC cell lines and immortalized CAFs. (B) Representative images of

Alexa-488 labeled methionine analog incorporation in DMSO control, 100nM SVC112, and 100nM HHT

treated 013C cells. 100x magnification, Scale bars = 200µm. (C) Relative levels of CSC related proteins in

“normal”, CAF, and HNSCC cell lines. (D) Relative eEF2 protein levels across cancer and CAF cell lines

(graph of three independent western blot experiments). (E) eEF2 protein levels following SVC112 (100nM)

treatment. (F) Media transfer experiment to assess the ability of SVC112 (100nM) to inhibit protein synthesis

(Click-iT assay). Media containing SVC112 that was incubated alone (24h-media) or with cells (24h-cells)

inhibited proliferation of 013C and 036C cells similar to fresh media containing drug, confirming SVC112

stability. (G) Media transferred to measure the ability of SVC112 (100nM) to inhibit cell proliferation (SRB

assay). (H) Media (100nM SVC112) transferred following a 24h incubation suppressed Myc and Cyclin D1

levels as readily as fresh media. Cells clearly have mechanisms to recover protein synthesis even in the presence

of drug, which appears to be cell and protein specific. CAF=cancer associated fibroblast, SVC=SVC112,

HHT=homoharringtonine, HNSCC=head and neck squamous cell carcinoma.

10

Supplementary Figure 5. Reversibility of treatment and duration of response to SVC112. (A) Myc and

Cyclin D1 are suppressed following a 6h and 24h SVC112 treatments. Both Myc and Cyclin D1 levels return

(6h:24h) following a 6h SVC112 treatment, washing cells out of drug, and 18h in fresh media. (B) SVC112

decreased colony formation for 013C (n=3), 036C (n=6), 049C (n=6), and 067C (n=6). HNSCC cells that were 11

seeded at multiple time points after washing cells from drug and the addition of fresh media. (C) Decreased

colony formation for 067C (n=6) cells treated with SVC112 for 72h prior to washout and addition of fresh

media. (D) Decreased sphere formation of viable 067C (n=4) cells seeded at multiple times was still sustained

24h after SVC112 washout. (E) Individual cell line comparison of cancer related protein inhibition by 100nM

SVC112 and 100nM HHT following a 6h drug exposure. (F) Densitometry analysis of Myc and Cyclin D1

levels following 6h exposure to SVC112 or HHT relative to DMSO treated controls. A minimum of three

experiments were analyzed for each cell line. Dashed line represents baseline (DMSO control) protein levels for

each individual cell line. Graphed results are presented as mean ±SD. Statistical significance was calculated by

two-tailed Student’s t test (* P≤ .05, ** P≤ .01). HNSCC=head and neck squamous cell carcinoma,

SVC=SVC112, HHT=homoharringtonine.

12

Supplementary Figure 6. Analysis of SVC112 resistance in HNSCC cell lines. (A) Proliferation (SRB assay)

and colony formation (clonogenic assay) following radiation (0, 2, 4, 6, 8, 10Gy) for DMSO control and

SVC112 resistant cells. (B) Changes in protein levels in 013C and 036C SVC112 resistant cells lines (compared

to DMSO treated controls) that were chronically treated or washed out of drug 7 days before analysis. (C)

Densitometry analysis of protein expression for 013C and 036C SVC112 resistant cell lines. Graphed results are

13

presented as mean ±SD. Statistical significance was calculated by two-tailed Student’s t test (* P≤ .05, **

P≤ .01). HNSCC=head and neck squamous cell carcinoma.

14

Supplementary Figure 7. Effects of Sox2 expression on treatment and augmentation of radiation induced

checkpoint delays by SVC112. (A) Changes in sphere formation following forced expression of Sox2 in

HNSCC cell lines compared to Empty vector controls. (B) Changes in total protein synthesis (Click-iT assay)

following forced expression of Sox2 in HNSCC cell lines. (C) Cell cycle analysis of 013C and 036C cell lines 15

expressing Sox2 or Empty vector treated with radiation (4Gy), SVC112 (100nM) or combination radiation +

SVC112. Change in the fraction of cells in each phase of the cell cycle following treatment, compared to the

untreated (baseline) control. (D) Fraction of cells in each phase of the cell cycle following treatment with

SVC112 ± 4Gy radiation. (E) Cell cycle analysis every 6h for 48h of 013C and 036C cell lines treated with

radiation (4Gy), SVC112 (100nM) or combination radiation + SVC112. (Top) Change in the fraction of cells in

each phase of the cell cycle following treatment, compared to the untreated (baseline) control. (Bottom)

Representative cell cycle histograms at 12h and 24h following treatment. Graphed results are presented as mean

±SD of three independent experiments. Statistical significance was calculated by two-tailed Student’s t test (*

P≤ .05, ** P≤ .01). HNSCC=head and neck squamous cell carcinoma.

16

SUPPLEMENTARY TABLES

Supplementary Table 1. Expected versus observed clonogenic survival for different dosing schedules of

radiation (4Gy) and SVC112.

4Gy + SVC112 (nM) 5 10 15 20

Expected survival, if additive 83.1 60.9 43.0 25.9

Observed survival, when drug was added

24h before 4Gy 90.2 (109%)n.s.

61.2 (100%)n.s.

40.6 (94%)n.s.

23.0 (89%)n.s.

Immediate after 60.2 (72%)P<.001

44.9 (74%)P<.001

21.6 (50%)P<.001

16.6 (64%)P<.05

24h after 4Gy 66.9 (81%)P<.001

47.4 (78%)P<.001

30.7 (71%)P<.001

23.4 (90%) n.s.

Tabulated clonogenic survival data from (Supplementary Figure 1B). Expected survival was computed as a

product of each single agent acting in an additive manner. Observed survival was expressed as the percentage of

expected and shown in brackets. Significance was calculated in EXCEL using the function

p=NORM.DIST(x,mean,STD,cumulative) where x = average survival at the schedule being tested, mean =

expected survival if additive, STD = standard deviation of the of the expected survival, and cumulative =

TRUE, for two-tailed distributions. The addition of drug immediately after radiation produced the lowest

clonogenic survival.

17

Supplementary Table 2. Characteristics of PDX tumors treated with radiation +/- SVC112.

Case TP53 HPV TNM PDX Tissue Primary Site

CUHN004 Mutant Negative T3N1M0 Local relapse Anterior Tongue

CUHN013 Mutant Negative T3N2cM0 Primary Site Submental Mass

CUHN036 Mutant Negative T4N2bM0 Primary Site Hypopharynx

CUHN047 WT Positive T1N2bM0 Lymph Node Tonsil

PDX=patient-derived xenograft, HPV=human papilloma virus, WT= wild type.

18

Supplementary Table 3. Incidence of histopathology in the organs of SVC112 treated mice.

Treatment esophagus trachea thymus lungs heart

Vehicle 0/3 0/3 0/3 0/3 0/3

SVC112 0/3 0/3 0/1* 0/3 0/3Abnormal/total number of organs examined in organs harvested from Balb/C mice treated with vehicle or

SVC112 for 4 weeks. Body weight fluctuation was insignificant (<10%) within the 4 weeks of treatment. Organ

weights were measured to address edema and did not correlate with SVC112 treatment (data not shown).

*absent after dissection.

19

Supplementary Table 4. GSEA results for RNA-seq analysis of HNSCC cells (013C, 049C, 067C) treated

with SVC112 for 6h.

HALLMARK PATHWAY ES P-value* FDR q-valueTNFα Signaling via NF-κB 0.774 <0.001 <0.001Inflammatory Response 0.503 <0.001 <0.001IL6 JAK Stat3 Signaling 0.542 <0.001 <0.001Apoptosis 0.481 <0.001 <0.001p53 Pathway 0.447 <0.001 <0.001Hypoxia 0.436 <0.001 <0.001KRAS Signaling Up 0.430 <0.001 <0.001Interferon Gamma Response 0.418 <0.001 0.001TGF-β Signaling 0.487 0.006 0.004Epithelial to Mesenchymal Transition 0.378 <0.001 0.007UV Response Up 0.383 <0.001 0.011UV Response Down 0.376 0.004 0.016Interferon Alpha Response 0.391 0.016 0.025Heme Metabolism 0.348 0.004 0.023Complement 0.350 0.008 0.024Reactive Oxygen Species Pathway 0.437 0.040 0.027Protein Excretion 0.383 0.009 0.027Myc Targets (V2) -0.343 0.079 0.189Wnt β-Catenin Signaling -0.382 0.051 0.159Angiogenesis -0.521 0.003 0.013

Positive ES increase with treatment while negative ES is a decrease in expression. *Statistical significance of

GSEA data was calculated by a two-sided modified Kolmogorov-Smirnov permutation test. GSEA=gene set

enrichment analysis, HNSCC=head and neck squamous cell carcinoma, ES=enrichment score.

20

SUPPLEMENTARY REFERENCES

1. Gladstone M, Frederick B, Zheng D, Edwards A, Yoon P, Stickel S, et al. A translation inhibitor

identified in a Drosophila screen enhances the effect of ionizing radiation and taxol in mammalian

models of cancer. Dis Model Mech 2012;5:342-50

2. Hall EJ, Giaccia AJ. Radiobiology for the Radiologist. 530 Walnut Street, Philadelphia, PA 19106

USA2006.

21