D E F
DMSO VLS-101 0
50 100
150
JeKo-1
% C el l C ycl e
0.01 0.1 1 10 100
0 10 20 30 40 50 60 70 80 90 100
VLS-101 (µg/ml)
% Cell Viability
JeKo-1
JeKo BTK KD_2 Mino
Z138
A B
JVM13 JVM
2Z138
JeKo R SP49 JeKo BTK KD_
1 JeKo-1 JeKo BTK KD_
2 Rec-
1 Mave
r Mino 0
10000 20000 30000
Fl uor es ce nc e u ni t
IsotypeROR1
C
JeKo
JeKo BTK KD_
2 Mino Z138
0.0 0.5 1.0 1.5
% Cell viability
0µg/ml 0.33µg/ml 3µg/ml 9µg/ml
P=0.0003 P=0.0001
P=0.00005 P=0.00001 P=0.000004
P=0.01
P=0.0001 P=0.00001
P=0.0005
0h 24h 72h 0.0
0.5 1.0 1.5
JeKo-1
% C ell v ia bilit y
P=0.0001P=0.00005
JeKo-1 JeKo BTK KD_2
0 20 40 60 80
% Apoptotic Cells
DMSO VLS-101
P=0.0001
P=0.000001
DMSO VLS-101 0
50 100
150
JeKo BTK KD_2
G0/G1 S G2/M
G
JeKo-1 Mino JVM2 JVM13 40
60 80 100 120
% C ell v ia bilit y
0 h 24 h
Supplementary Figure S1
Supplementary Figure S2
PDX-1 PDX-2
PDX-3 PDX-4
PDX-5 PDX-6
PDX-7 PDX-8
PDX-9 PDX-10
0
5000 10000 15000
Fl uor es ce nc e u ni t Isotype ROR1
Patient PDX 0
2000 4000 6000
Fl uor es ce nc e u ni t p = 0.76
A B
Vehicle VLS-101 1000
2000 3000 4000
Li ve r w ei gh t ( m g) P=0.0020
Vehicle VLS-1010 200 400 600 800
S pl ee n w ei ght (m g) P=0.0001
B
VLS-101 - + - +
Spleen Liver
#1
#2
#3
#4
#5
A
Vehicle VLS-101 0
50 100 150
% CD5
+CD2 0
+cel ls i n sp lee n
P=3.85e-17
Vehicle VLS-101 0
50 100 150
% CD5
+CD2 0
+c ells in liv er
P=7.58e-6
C
Supplementary Figure S3
Supplementary Figures
Figure S1. ROR1 expression and ROR1 targeting in MCL cell lines. (A) ROR1 expression detected by flow cytometry in MCL cell lines (n = 11). (B) In vitro efficacy of VLS-101 in 4 representative MCL cell lines, JeKo-1, JeKo-BTK-KD, Mino and Z138 at 72 h post treatment with VLS-101. (C) Dose-dependent inhibition of cell viability by VLS-101 at 24 h post treatment in MCL cell lines. (D) Time-dependent inhibition of cell viability by VLS-101 treatment in JeKo-1 cells. (E) ROR1 expression dependent cell viability inhibition at 24 h post treatment. Note the lack of VLS-101 inhibitory effect in ROR1-negative cells (JVM2 and JVM13). (F-G) Cell apoptosis (F) and cell cycle arrest (G) induction by VLS-101 at 24 h post treatment in ibrutinib-sensitive (JeKo-1) and resistant (JeKo BTK KD_2) cell lines. Student t test was used to calculate statistical significance.
Figure S2. ROR1 expression in PDX models and their parental patient samples. (A) ROR1 expression detected by flow cytometry in MCL PDX models (n = 10). (B) Comparison of ROR1 expression levels between parental clinical samples and PDX models using flow cytometry (n = 5).
Figure S3. VLS-101 is potent in targeting MCL cells in ROR1-expressing PDX models with dual
resistance to ibrutinib and venetoclax. A PDX model was established from an ibrutinib-venetoclax dual resistant MCL patient tumor using a multi-generational expansion in immunodeficient NSG mice. Fresh isolated PDX cells from the spleen of previous mouse generation were inoculated intravenously into NSG mice. At 2 weeks post cell inoculation, the mice were treated intravenously with vehicle (n = 5) or VLS- 101 (n = 5) at 2.5 mg/kg (QW x 3). (A-C) At 8 weeks post cell inoculation, the mouse spleen (left panel) and liver (right panel) were dissected, imaged (A) and weighted (B). The percentage of MCL cells in the spleen (left panel) and liver (right panel) were detected by flow cytometry with fluorescence conjugated CD5 and CD20 antibodies (C). Student t test was used to calculate statistical significance.
