1
Supplementary Information for Disruption of IRE1a through its Kinase Domain Attenuates Multiple Myeloma Jonathan M. Harnossa, Adrien Le Thomasa, Anna Shemorrya, Scot A. Marstersa,
David A. Lawrencea, Min Lua,1, Yung-Chia Ariel Chena,2, Jing Qinga, Klara Totpalb,
David Kanb, Ehud Segalb, Mark Merchantb, Mike Reicheltc, Heidi Ackerly Wallweberd,
Weiru Wangd, Kevin Clarke, Susan Kaufmane, Maureen H. Beresinie, Steven T. Laingc,
Wendy Sandovalf, Maria Lorenzog, Jiansheng Wug, Justin Lyh, Tom De Bruynh, Amy
Heidersbachi, Benjamin Haleyi, Alvin Goginenij, Robby M. Weimerj, Dong Leek,2,
Marie-Gabrielle Braunl, Joachim Rudolphl, Michael J. VanWyngardenm, Daniel W.
Sherbenoum, Patricia Gomez-Bougien,o, Martine Amiotn,o, Diego Acosta-Alvearp,q,3,
Peter Walterp,q and Avi Ashkenazia,4
4Corresponding author: Tel: +1 650-225-1853, email: [email protected] This PDF file includes:
Supplemental Material and Methods Figs. S1 to S6 including Fig. legends References for SI appendix
www.pnas.org/cgi/doi/10.1073/pnas.1906999116
2
Supplemental Material and Methods
Generation of shRNA-expressing cells. Cells were infected with shRNAs by lentivirus
using the pG-GW-pHUSH vector system. Briefly 1x107 293T cells were plated in 6-well
dishes, allowed to grow for 24 hours and transfected with lentivirus plasmids using
Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol. After 6 hours the
media was replaced, and after another 24 hours the virus was harvested from the cells and
filtered through a 0.45 mm tube-top filter. Cleared supernatants containing the viral
particles and 8 mg/ml polybrene were added to the target-cell wells, spun at 1800 rpm for
45 min at room temperature, and placed back into the incubator. After 48 hours cells
were subjected to selection with puromycin containing media. After 10 passages, cells
were tested for virus by Lentivirus X p24 Rapid Titer Kit (Clonetech) then sorted using
FACS and RFP+ selection.
NTC shRNA sequence (antisense): TAGATAAGCATTATAATTCCT
IRE1a shRNA sequences (antisense):
shRNA5: AGCTTTTCCAAAAAACCAAGATGCTGGAGAGATTTCTCTTGATAATCTCTCCAGCATCTTGGGGG
shRNA7: AGCTTTTCCAAAAAAAGAACAAGCTCAACTACTTTCTCTTGATAAGTAGTTGAGCTTGTTCTGGG
shRNA8: AGCTTTTCCAAAAAAGCACGTGAATTGATAGAGATCTCTTGAATCTCTATCAATTCACGTGCGGG
shRNA9: AGCTTTTCCAAAAAAGAGAAGATGATTGCGATGGTCTCTTGAACCATCGCAATCATCTTCTCGGG
XBP1s shRNA sequences (antisense):
3
shRNA6: AGCTTTTCCAAAAAAGCAGGTAGATTTAGAATCTCTTGAATTCTAAATCTACCACTTGCGGG
shRNA: AGCTTTTCCAAAAAAGCTGGAAGCCATTAATGAATCTCTTGAATCTCTTGAATTCATTAATGGCTTCCAGCGGG
CRISPR/Cas9 knockout of IRE1a and XBP1 genes. Individual IRE1a- or XBP1-
specific sgRNAs were designed using a standard guide scaffold and CRISPR3 (1, 2). The
gRNAs were cloned into pLKO_AIO_CMV_Cas9_mCherry, enabling co-expression of
each sgRNA, Cas9, and an mCherry-based selection marker following transient
transfection into target cells. A tandem array of XBP1-specific sgRNAs were designed as
above and cloned into pLKO_AIO_TAN_PGK_Cas9_Puro, permitting co-expression of
two XBP1-targeting sgRNAs, Cas9, and a puromycin selection marker in transfected
cells.
sgRNA target sequences used in this study:
IRE1a gRNA1: TCAGGAAGCGTCACTGTGC
IRE1a gRNA2: GAGGACAGGCTCAATCAAA
IRE1a gRNA3: TTCTCCCAGATCCTAATGA
XBP1 gRNA1: TTTAGGGGTCCCGTCGGCC
Transfection was with Lipofectamine 3000 (Invitrogen) according to manufacturer's
protocol. At 24 hours after transfection, cells were washed once in PBS and resuspended
in PBS media containing 3% BSA Fraction V. The cell suspension was then filtered
through a 35 mm membrane followed by immediate FACS sorting using the RFP+
selection marker. Single cell clones (n=96) were plated and grown. Clones producing
colonies were tested for proper IRE1a or XBP1 disruption by immunoblot.
Reconstitution experiments. For the in vitro reconstitution experiments, a plasmid
containing the cDNA sequence for wild-type, kinase-dead (D688N), or RNase-dead
4
(K907A) IRE1a under the control of a CMV promoter was transiently transfected into
the KMS-11 IRE1a CRISPR KO Cl. 2.3 with Lipofectamine 3000 according to the
manufacturer's protocol. After 2 days, cells were treated with either DMSO or 100 nM Tg
for 3 hours. Cells were harvested and protein lysates were analyzed by immunoblot.
For the in vivo reconstitution experiments, a plasmid containing the cDNA sequence for
either wild-type or kinase-dead (D688N) IRE1a under the control of the endogenous
IRE1a promoter was transfected into KMS-11 IRE1a KO Cl. 2.3 with either
Lipofectamine 2000 or 3000 according to the manufacturer's protocol. Transfected cells
were selected with neomycin (600 µg/ml) to generate stable cell lines and clones were
isolated.
RT-qPCR. RNA was extracted with RNeasy Plus kit (Qiagen). Equal amounts of RNA
were reverse transcribed and amplified by RNA-to-CT kit (Applied Biosystems). The
delta-delta CT values were calculated by relating each individual CT value to its internal
GAPDH control, and then normalizing to the vehicle-treatment control. RNA was
purified from cells using the RNeasy Plus kit (Qiagen). Equal amounts of RNA were
reverse transcribed and amplified by RNA-to-CT kit (Applied Biosystems) on the ABI
QuantStudio 7 Flex Real-Time PCR System. The delta-delta CT values were calculated
by relating each individual CT value to its internal GAPDH control, and then normalized
to the vehicle-treatment control.
Taqman primers (Life Technologies):
GAPDH: Hs02758991_g1
XBP1u: Hs02856596_m1
XBP1s: Hs03929085_g1
DGAT2: Hs01045913_m1
SYVN1: HS00381211_m1
DNAJC10:Hs00405977_m1
DERL2: Hs00211351_m1
ERLEC1: Hs01048033_m1
UBE2J1: Hs00249272_m1
5
EDEM1: HS00976004_m1
VIMP: Hs00218369_m1
In vitro characterization of small molecule inhibitors. Small molecule potencies were
assessed in three assays of IRE1a function. Compound dilutions covering a range of
concentrations from 0.2 nM to 10 μM were evaluated to determine IC50 values.
Compound binding to the IRE1a ATP site was assessed through competition with an
Alexa647-labeled staurosporine probe for binding to His-tagged IRE1a (G547-L977
D688N). Probe binding was measured as TR-FRET signal upon energy transfer between
the bound probe and anti-His-allophycocyanin bound to the IRE1a. To assess inhibition
of RNase activity, compound was mixed with IRE1a (Q470-L977), and 5’FAM-
CAUGUCCGCAGCGCAUG-3’BHQ substrate was added. Substrate cleavage was
monitored kinetically as an increase in fluorescence. Cellular activity was evaluated via
XBP1s-luciferase reporter assay. HEK293T cells stably transfected with the reporter
construct were preincubated with compound for 2 hours and then stimulated with Tg (100
nM) for 6 hours. IRE1a-mediated cleavage and splicing of the reporter construct led to
expression of luciferase, which was detected by the addition of luciferin substrate.
Immunoblot analysis. Cells were lysed or tumor tissues mechanically disrupted in 1x
RIPA buffer (Millipore) containing protease and phosphatase inhibitors (Roche), cleared
by centrifugation at 13,000 rpm for 10 min, and analyzed by BCA protein assay
(Thermofisher Scientific). Equal protein amounts were loaded, separated by SDS-PAGE,
electro-transferred to nitrocellulose membranes using the iBLOT2 system (Invitrogen),
and blocked in 5% nonfat milk solution for 2 hours. Membranes were probed with the
following antibodies: IRE1a, GAPDH, b-actin (Cell Signaling Technology), XBP1s,
phosphorylated IRE1a (Genentech (3)). Signal was detected using appropriate
horseradish peroxidase (HRP)-conjugated secondary antibodies. All primary antibodies
were used at 1:000 dilution and overnight hybridization at 4°C, followed by a 1-hour
incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies at
1:10,000 dilution.
6
Luminex and ELISA analysis. For in vitro analysis of secreted chemokines and
cytokines, RPMI-8226 NTC shRNA and IRE1a sh7-5 cells were incubated in the
absence or presence of Dox (1 µg/ml) for 3 days, an equal number of cells seeded (1x106/
well), and cell supernatants analyzed after 48 hours cells by Luminex Premix Panel I 29-
plex (Biorad). Concentration of analyzed chemokines and cytokines was normalized to
cell number after 48h. For in vivo analysis of secreted chemokines and cytokines, sera
from tumor-bearing mice were analyzed using Luminex Premix Panel I 29-plex. For
analysis of light-chain secretion, equal number of KMS11 WT or IRE1a KO Cl. 2.3 cells
were seeded (0.25x106). RPMI-8226 NTC shRNA and IRE1a sh7-5 cells were incubated
in the absence or presence of Dox (1 µg/ml) for 3 days, an equal number of cells seeded
(1.5x106/ well), and cell supernatants were analyzed after 9 hours for IgG k or l light-
chain secretion, respectively, using a human-specific light chain antigen capture enzyme-
linked immunosorbent assays (ELISA) (Abcam). Pancreatic islet 3D microtissues were
incubated for 7 days with serial dilutions of compound 18 or vehicle control (DMSO),
and insulin secretion analyzed after glucose challenge for 1 hour (16.7 mM) by ELISA
(Mercodia).
Co-crystallographic studies. The kinase-RNase (KR) domain of hIRE1a, encoding
amino acids G547-L977, was expressed as an N-terminal His6-tagged fusion protein in
SF9 cells with a TEV protease cleavage site from an intracellular BEVS expression
vector. Cell pellet was resuspended in lysis buffer containing 50 mM HEPES pH 8.0, 300
mM NaCl, 10% glycerol, 1mM MgCl2, 1:1000 benzonase, EDTA-free PI tablets (Roche),
1mM TCEP, and 5mM imidazole. Sample was homogenized, compound 18 was added to
a final concentration of 10 µM, and incubated at 4°C for 30 min. Sample was lysed by
sonication, ultracentrifuged at 40,000 rpm for 45 min, and the supernatant filtered
through a 0.45 µ Nalgene filter. Cleared supernatant was batch bound to Ni-NTA
Superflow beads (Qiagen) for 1 hour at 4°C with nutation. Beads were washed in lysis
buffer supplemented with 25 mM imidazole, followed by protein elution in lysis buffer
containing 300 mM imidazole. The eluate was concentrated and loaded onto a Hiload
16/600 Superdex 75 SEC column (GE Healthcare) equilibrated in 25 mM HEPES pH 7.8,
7
250 mM NaCl, 10% glycerol, 1 mM TCEP (SEC buffer). The monomeric peak was
pooled and treated with lambda phosphatase for 1 hour at 37°C. Dephosphorylation to 0P
was confirmed by mass spectrometry. Sample was incubated with TEV protease
overnight at 4°C for tag removal. Untagged protein was isolated by passage over a Ni-
NTA Superflow gravity column, followed by a wash with SEC buffer supplemented with
40mM imidazole. Flow-through and wash fractions were pooled, diluted 1:10 in Q buffer
(25 mM HEPES pH 7.8 and 1 mM TCEP), and loaded onto a 5 ml prepacked QHP
column (GE Healthcare). Protein was eluted over a 50 CV gradient in Q buffers
supplemented with 25 mM and 500 mM NaCl. The hIRE1a_KR + 18 complex peak was
isolated and concentrated to 9.4 mg/ml for crystallography. Crystals were generated in
hanging drops with mother liquor containing 0.1 M trisodium citrate pH 5.6, 10%
isopropanol, 10% PEG4000, and cesium chloride additive at 4°C. Crystals were
cryoprotected in the crystallography buffer supplemented with 25% glycerol and flash
frozen in liquid nitrogen. Data collection was done at ALS 5.0.2. Structure was solved to
2.2 Å.
BSE-SEM studies. Subcutaneous KMS11 tumor xenografts (parental, IRE1a KO Cl. 1.1
or 2.3, n=2/ group) were harvested and fixed by immersion fixation in modified
Karnovski's fixative (2.5% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2) for at least 24 hours or longer at 4 °C. Fixed tissues slices were washed
with ultrapure water and post-fixed with 1% osmium tetroxide reduced with 1.5% (w/v)
potassium ferrocyanide (final concentration) for 2 hours on ice. The samples were then
washed again in ultrapure water and stained “en block” with 0.5% (w/v) uranyl acetate at
4 °C overnight. Following staining, samples were dehydrated in a series of ascending
ethanol concentrations, rinsed twice with propylene oxide and finally embedded in epoxy
resin Eponate-12 (Ted Pella).
Clean "superfrost-plus" microscope slides (Thermo Fisher Scientific) were sputter
coated with a layer of about 40-80 nm carbon from carbon-cord resulting in a surface with
shiny brown color, using a sputter coater (EMS150R ES sputter coater, Electron
Microscopy Sciences). Thick sections (1000 nm thickness) were cut with the UMC
ultramicrotome (Leica Biosystems) using a DIATOME diamond knife for histology
8
(Electron Microscopy Sciences). Sections were transferred to the carbon-coated glass
slides and dried. Finally, sections were stained with 4% aqueous uranyl acetate for 15 min
and 0.1% Reynold’s lead citrate (4) for 1 min to enhance contrast. Sections were
thoroughly rinsed with water and dried on a heat plate before being transferred to the SEM.
Backscattered electron scanning electron microscopy (BSE-SEM) was performed
using a GeminiSEM 300 equipped with a field emission gun (Carl Zeiss AG). For operation
of the GeminiSEM 300 microscope the application software SmartSEM (version 6.01) was
used (Carl Zeiss AG). Imaging was with the backscatter electron detector (BSD1) at 8.5
mm working distance, 30 µm (standard) aperture, 3-6keV acceleration voltage and with
operation of the field emission gun in "high current" mode. For the majority of images, a
scan speed of "5", noise reduction by 4x line averaging and an image size of at least 4096
x 3072 (4k x 3k) pixels was chosen. For imaging of ultrastructural detail pixel sizes
between 2-5 nm were used. The greyscale of the images was inverted to achieve TEM-like
representations.
Photoshop CS4 (Adobe) was used to adjust contrast and brightness of whole
images, to crop regions of interest and to reduce the pixel size per area for images that were
obtained with a store resolution larger than 4096 x 3072 pixels to produce images at 300
dots per inch (dpi) print resolution for figure preparation.
In vivo pharmacokinetic analysis. Pharmacokinetic properties of compound 18 were
determined in 5 to 6 weeks old female C.B-17 SCID mice (Charles River Laboratories)
bearing subcutaneous KMS-11 tumor xenografts (3 mice/ group). Mice were treated
intraperitoneally (IP) with compound 18 (30 mg/kg, formulated in 35% PEG400 and 10%
EtOH in water) either as a single dose (QD), or twice per day (BID) over 4 days. Food
and water were available ad libitum to all animals. Serial blood samples (15 µL) were
collected by tail nick at 0.25, 0.5, 1, 2, 4, 6, or 8 hours after the last 18 injection. Blood
samples were diluted with 60 µL water containing 1.7 mg/mL EDTA and kept at −80°C
until analysis. Plasma concentrations of 18 were determined by a non-GLP LC/MS-MS
assay.
9
Compound 18 tolerability studies. The tolerability study with compound 18 dosed BID
was done in 5 to 6 weeks old female C.B-17 SCID mice (Charles River Laboratories).
Mice (n=4 animals/ group) were injected 18 at 0, 10, 30, or 100 mg/kg, 100 µL total, IP,
BID for 7 days. On day 8 blood was drawn and processed routinely for hematology and
clinical chemistry analysis, and animals were euthanized and subject to a full gross
necropsy. Selected tissues (SI Appendix, Fig. S6C) were fixed in neutral buffered
formalin for a minimum of 24 hours prior to being processed, embedded in paraffin,
sectioned at 5 µm and stained with hematoxylin and eosin. Hematology, clinical
chemistry, organ weight data (absolute and relative to body weight or brain weight), and
tissue sections were assessed by a board-certified veterinary pathologist (STL). The
tolerability study with 18 dosed QD was done in 7 to 8 weeks old NOD/SCID/IL2rγ-/-
mice (NSG, Jackson Laboratories). Mice (n=12 in vehicle, n=13 in Compound 18 group)
were administered 18 at 30 mg/kg, 100 µL total, IP, QD for 21 days. On day 21 blood
was drawn and processed routinely for clinical chemistry analysis.
In vivo xenograft tumor growth studies. Tumor size and body weight were measured
twice per week. Subcutaneous tumor volumes were measured in two dimensions (length
and width) using Ultra Cal-IV calipers (model 54 − 10 − 111; Fred V. Fowler Co.) and
analyzed using Excel, version 11.2 (Microsoft), or Prism 6 (GraphPad Software, Inc.).
The tumor volume was calculated with the following formula: tumor size (mm3) =
(longer measurement × shorter measurement2) × 0.5. Animal body weights were
measured using an Adventurer Pro AV812 scale (Ohaus Corporation). Percent weight
change was calculated using the following formula: group percent weight change = [(new
weight − initial weight)/initial weight] × 100. To analyze the repeated measurement of
tumor volumes from the same animals over time, a mixed modeling approach was used
(5). This approach addresses both repeated measurements and modest dropouts before the
end of study. Cubic regression splines were used to fit a nonlinear profile to the time
courses of log2 tumor volume in each group. Fitting was done via a linear mixed-effects
model, using the package "nlme" (version 3.1-108) in R version 2.15.2 (R Development
Core Team 2008; R Foundation for Statistical Computing; Vienna, Austria). Tumor
growth inhibition (TGI) as a percentage of vehicle was calculated as the percentage of the
10
area under the fitted tumor volume–time curve (AUC) per day for each treatment group
in relation to the vehicle control using the following formula: %TGI = %TGI = (1-
[(AUC/Day)Treatment ÷ (AUC/Day)Vehicle]) × 100. When mice reached endpoint criteria (see
below) or on the last treatment day, mice were euthanized by cervical dislocation and
subcutaneous xenografts harvested for immunoblot analysis.
Animals in all studies were humanely euthanized according to the following
criteria: clinical signs of persistent distress or pain, significant body-weight loss (>20%),
tumor size exceeding 2500 mm3, or when tumors ulcerated. Maximum tumor size
permitted by the Institutional Animal Care and Use Committee (IACUC) is
3000 mm3 and in none of the experiments was this limit exceeded.
11
KMS-11 IRE1a sh8-9
- + Dox:
Day
0Da
y 5
Multiple
myeloma
Medullo
blastoma
T-cell_
ALL
Prosta
te
Lymphoma_
Burkitt
Bile_d
uctCML
Pancre
as NA
B-cell_
lymphoma_
other
Colorectal
AML
Stomach
Lung_NSCOther
Upper_ae
rodiges
tive
Esophag
us
Lung_small
_cell
Mesotheli
omaLive
r
T-cell_
lymphoma_
others
Urinary
_trac
t
Breas
t
EndometriumOva
ry
Soft_tis
sue
Thyroid
Melanoma
Leuce
mia_other
Lymphoma_
DLBCL
B-Cell
_ALL
Neuro
blastoma
Lymphoma_
Hodgkin
Meningioma
Giant_c
ell_tu
mor
Ewings_Sarc
oma
Chondrosa
rcoma
Kidney
Glioma
Osteosa
rcoma
-2
0
2
4
6
8
rpkm
ERN1 mRNA expression A
- Dox
+ Dox
0
2×107
4×107
Lum
ines
cenc
e (R
LU)
KMS-11 WT
- Dox
+ Dox
0.00
0.75
1.50
XBP1
s (fo
ld c
hang
e to
bas
elin
e)
KMS-11 IRE1α sh8-9
**
- Dox
+ Dox
0.0
1.5×107
3.0×107
Lum
ines
cenc
e (R
LU)
KMS-11 IRE1α sh8-9
***
0 50 100 1500
5×105
1×106
Hours
Obj
ect a
rea
(um
2 )
- Dox+ Dox
KMS-11 IRE1α sh8-9D
0 50 100 1500
5×105
1×106
Hours
Obj
ect a
rea
(um
2 )
- Dox+ Dox
KMS-11 WTB
HG
E
CKMS-11 WT
-Dox: +
Day
0Da
y 5
F
Fig. S1
12
Fig. S1 Expression of IRE1a in cancer cell lines and effect of its shRNA-based
depletion on 3D spheroid growth of KMS-11 MM cells. (A) The cancer cell line
encyclopedia (CCLE) dataset, which comprises RNAseq-based analysis of human cancer
cell lines including 29 MM lines (Broad Institute, Cambridge, MA, USA)
(https://portals.broadinstitute.org/ccle/page?gene=ERN1) was queried for expression of
IRE1a (ERN1). (B-E) KMS-11 parental IRE1a wildtype (WT) cells (B and C) or cells
stably transfected with plasmids encoding doxycycline (Dox)-inducible shRNAs against
IRE1a (D and E) were treated with Dox (0.5 µg/ml) for 3 days, seeded on Matrigel,
allowed to grow as multiple 3D-spheroids, and analyzed over 5 days in an IncucyteTM S3
instrument. Representative images for KMS-11 WT (C) and KMS-11 IRE1a sh8-9 cells
(E) grown on Matrigel. (F-H) Cells were treated as in B and C and analyzed by
CellTiter-Glo® 3D to determine cell viability (F and H) or by RT-qPCR to determine fold
change of XBP1s mRNA levels (G). Scale bars = 400 µm. **p≤0.01, ***p≤0.001.
13
0 5 10 15 20 250
1750
3500
Dox + lenalidomide
Day
Tum
or v
olum
e (m
m3 )
0 5 10 15 20 250
1000
2000
Dox + bortezomib
Day
Tum
or v
olum
e (m
m3 )
+ WT RIRE1a Cl. 1
KMS-11 KMS-11 xenograftsB CA KMS-11 xenografts
GAPDH
IRE1a
XBP1s
ParentalCl. 1.1
Cl. 2.3
Cl. 3.1
IRE1a KO
GAPDH
Parental Cl. 1.14 Cl. 1.8
IRE1a
XBP1s
KMS-11 xenografts
XBP1s KO
IRE1a
XBP1s
GAPDH
Parental Cl. 2.3
KD RIRE1a
Cl. 1
KD RIRE1a
Cl. 5
KD RIRE1a
Cl. 8
IRE1aKO
KMS-11 xenografts E
I
0 5 10 15 20 250
1000
2000
Bortezomib
Day
Tum
or v
olum
e (m
m3 )
0 5 10 15 20 250
1750
3500
Lenalidomide
Day
Tum
or v
olum
e (m
m3 )
0 5 10 15 20 250
1750
3500
Day
Tum
or v
olum
e (m
m3 )
Vehicle
0 5 10 15 20 250
1750
3500
Dox
Day
Tum
or v
olum
e (m
m3 )
0 5 10 15 20 250
1000
2000
Day
Tum
or v
olum
e (m
m3 )
Vehicle
0 5 10 15 20 250
1000
2000
Dox
Day
Tum
or v
olum
e (m
m3 )
HG
Treatment group
N of animals day 0/ last
day
AUC/ day % TGI mean
(range)
AUC/ day mean
(range)
1 Vehicle (5% sucrose) 10/9 0 (0, 0) 431
(281, 671)
2 Doxycyline(0.5 mg/mL, PO) 10/9 61 (24, 81) 167
(91, 289)
3Bortezomib(0.75 mg/kg IV BIW)
10/10 54 (7, 79) 197 (108, 328)
4
Bortezomib(0.75 mg/kg IP BIW) +Doxycyline(0.5 mg/mL, PO)
10/9 91 (73, 102) 40 (-8, 104)
Treatment group
N of animals day 0/ last
day
AUC/ day % TGI mean
(range)
AUC/ day mean
(range)
1 Vehicle (5% sucrose) 9/7 0 (0, 0) 619
(392, 903)
2 Doxycyline(0.5 mg/mL, PO) 8/8 70 (42, 84) 187
(109, 299)
3Revlimid(50 mg/kg IP QDx5)
8/8 61 (24, 78) 243 (140, 384)
4
Revlimid(50 mg/kg IP QDx5) +Doxycyline(0.5 mg/mL, PO)
8/8 100 (92, 107) 0 (-41, 45)
shNTC
IRE1α sh
8-9
shNTC
IRE1α sh
7-50.0
0.5
1.0
1.5
Fold
cha
nge
to b
asel
ine - Dox
+ Dox
RPMI-8226KMS-11
Tumor volume
*****
KMS-11 IRE1a sh8-9 xenografts
- Dox + Dox
XBP1s
Actin
IRE1a
+ Dox
GAPDH
IRE1a
XBP1s
- Dox
RPMI-8226 IRE1a sh7-5xenografts
D F
J
Parental
XBP1s
Actin
Cl. 1.1 Cl. 2.3 Cl. 3.1
IRE1a
Tg (hr): 0 0 04 24 4 24 4 244 240
IRE1a KO
Parental Cl. 2.3
IRE1a
GAPDH
XBP1s
IRE1a KO
Fig. S2
14
RPMI-8226 IRE1α sh7-5
2 5 8 120.0
0.2
0.4
Hours
Free
IgG
λ li
ght c
hain
s (n
g/m
L)
- Dox+ Dox
N
- Dox
+ Dox
0.0
0.5
1.0
1.5
IP-10
Fold
cha
nge
to b
asel
ine
- Dox
+ Dox
0.0
0.5
1.0
1.5
IL-8
Fold
cha
nge
to b
asel
ine
- Dox
+ Dox
0.4
0.6
0.8
1.0
1.2
VEGF
Fold
cha
nge
to b
asel
ine
- Dox
+ Dox
0.0
0.5
1.0
1.5
IL-10
Fold
cha
nge
to b
asel
ine
- Dox
+ Dox
0.0
0.5
1.0
1.5
IL-1α
Fold
cha
nge
to b
asel
ine
- Dox
+ Dox
0.0
0.5
1.0
1.5
IL-6
Fold
cha
nge
to b
asel
ine
***
*** ***
***
IRE1a
XBP1s
GAPDH
Dox: - + - +
shNTC
shIRE1a
RPMI-8226
- Dox + Dox
RPMI-8226 IRE1a sh7-5
hr: 2 5 8 122 5 8 12
hIgG l
GAPDH
IRE1a
LK M
O
**
KMS-11 IRE1a KO Cl. 2.3KMS-11 WT KMS-11 IRE1a KO Cl. 1.1
N
N
N
Fig. S2
15
Fig. S2 Genetic disruption of IRE1a attenuates secretory function and growth of
subcutaneous human MM tumor xenografts in mice. (A) Parental and IRE1a KO
KMS-11 cells were treated with Thapsigargin (Tg, 100 nM) for the indicated time and
analyzed by IB for expression of indicated proteins. (B) IB analysis to confirm depletion
of IRE1a in corresponding subcutaneous WT and KO KMS-11 tumor xenografts. (C and
D) IB analysis to confirm reconstitution of WT IRE1a (WT RIRE1a, C) or kinase-dead
(KD) D688N mutant IRE1a (KD RIRE1a, D) in corresponding subcutaneous WT and
IRE1a KO Cl. 2.3 tumor xenografts. (E) IB analysis to confirm depletion of XBP1s in
corresponding subcutaneous WT and KO KMS-11 tumor xenografts. (F) KMS-11 or
RPMI-8226 cells stably transfected with Dox-inducible shRNAs against NTC or IRE1a
were inoculated subcutaneously into C.B-17 SCID mice and allowed to establish tumors
of ~200 mm3 in volume. After randomization into treatment groups (n=6-7 mice/ group
bearing KMS-11 tumors, 8-10 mice/ group bearing RPMI-8226 tumors), mice were
treated with either vehicle (sucrose) or Dox in drinking water, and tumor growth was
monitored over 21 days. Shown is the fold change of tumor volume on day 21 of
treatment. (G and H) Tumor growth trajectories of individual animals, corresponding to
the mean tumor volumes depicted in Fig. 2D (G) and Fig. 2E (H). (I and J) IB analysis
to confirm Dox-mediated shRNA depletion of IRE1a and XBP1s in individual KMS-11
(I) and RPMI-8226 (J) tumor xenografts. (K) RPMI-8226 cells containing NTC or
IRE1a shRNAs were cultured in the absence or presence of Dox (1 µg/ml) for 3 days and
analyzed by IB to confirm specific Dox-mediated depletion of IRE1a. (L and M) RPMI-
8226 cells harboring inducible IRE1a shRNAs were incubated in the absence or presence
of Dox (1 µg/ml) for up to 12 hours. Levels of human IgG l were analyzed in cell
supernatants by ELISA (L) or in cell lysates by IB (M). (N) RPMI-8226 cells harboring
inducible IRE1a shRNAs were incubated in the absence or presence of Dox (1 µg/ml) for
3 days, seeded at equal number, and the concentrations of indicated cytokines and
chemokines in the cell supernatants were analyzed by Luminex. Fold-change 48 hours
after seeding normalized to cell number is shown. (O) Representative BSE-SEM images
of KMS-11 parental or IRE1a KO tumor xenografts. Blue arrows indicate ER. N,
nucleus. *p<0.05, **p≤0.01, ***p≤0.001.
16
Vehicl
e 0.5 1 2 4 80
50
100
150
KMS-11
Gro
wth
(% o
f veh
icle
) Compound 18JNK-IN-8SP600125
µM
*** *** **** *** ***
A
B
Compound 18 +
IRE1a KRPDB code TBD
2016_09_14_ALS_502
/22744
_G02968219_KR_WT
Space group P212121
Unit cell a=67.1Å, b=84.7Å,
c=175.5Å,
α=β=γ=90°
Resolution 2.20 Å
Total reflections 367185 (3613) 1
Completeness (%) 100 (100)
Redundancy 7.1 (7.3)
FCompound 18 +
IRE1a KRI/σ 7.6 (2.8)
Rsym2 0.158 (0.670)
Resolution range 50-2.20 Å
Rcryst3 / Rfree4 0.220/0.274
Non-hydrogen atoms 7116
Water molecules 589
Average B 26.4 Å2
r.m.s.d. bond lengths 0.006 Å
r.m.s.d. angles 0.925°
Ramachandran 0.908/0.088/0.001/
0.003
C
Compound 18 16 KIRA6
IRE1 FRET assay IC50 (nM) 3.1 3.7 15.3
IRE1 RNase assay IC50 (nM) 3.3 4.2 79.7
XBP1 reporter cell assay IC50 (nM) 35 57 140
KinomeScanTM Invitrogen assay at 1 µMKinases with > 90% off-target inhibitionKinases with > 70-90% off-target inhibition
1/2200/220
2/2205/220
38/22026/220
Plasma protein binding (human, mouse, %) 99.4, 99.4 99.2, 99.7 ND
00.0
10.0
3 0.1 0.3 1 30
20
40
Compound 18 (µM)
XBP1
s (%
of t
otal
)
RPMI-8226
1 10 100 10000
4
8
0.50
0.75
1.00
Compound 18 (nM)
XBP1
s (A
U)
KMS-11
DGAT2 (AU)
IC50= 82.5 nM IC50= 76.5 nM
1.0 2.0 3.0 4.0 5.0 6.00
50
100
rpkm
Num
ber o
f can
cer c
ell l
ines
JNK2 mRNA expression
OPM-2
RPMI-8226
KMS-11
G
D E
Compound 18 KIRA6Compound 16
Fig. S3
17
Vehicl
e 1.9 3.8 7.5 15 300
50
100
150
RPMI-8226 IRE1α sh7-5
Gro
wth
(% o
f veh
icle
) 2D3D
4µ8c (µM)
*** ***
Vehicl
e 1.9 3.8 7.5 15 300
50
100
150
OPM-2 IRE1α sh9
Gro
wth
(% o
f veh
icle
) 2D3D
4µ8c (µM)
*** *** *** ***
Vehicl
e 1.9 3.8 7.5 15 300
50
100
150
KMS-11 IRE1α sh8-9
Gro
wth
(% o
f veh
icle
)
3D
4µ8c (µM)
2D*** *** ***
0 50 100 150 2000
10
20
30
RPMI-8226 IRE1α sh7-53D
Hours
Con
fluen
ce (%
)
Vehicle0.31 uM Compound 180.63 uM Compound 181.25 uM Compound 182.5 uM Compound 185 uM Compound 18
0 50 100 150 2000
10
20
30
OPM-2 IRE1α sh93D
Hours
Con
fluen
ce (%
)
Vehicle0.31 uM Compound 180.63 uM Compound 181.25 uM Compound 182.5 uM Compound 185 uM Compound 18
0 50 100 150 2000
10
20
30
40
HoursC
onflu
ence
(%)
KMS-11 IRE1α sh8-93D
Vehicle0.31 uM Compound 180.63 uM Compound 181.25 uM Compound 182.5 uM Compound 185 uM Compound 18
0 50 100 150 2000
20
40
60
80
KMS-11 IRE1α sh8-92D
Hours
Con
fluen
ce (%
)
Vehicle0.31 uM Compound 180.63 uM Compound 181.25 uM Compound 182.5 uM Compound 185 uM Compound 18
H
K
JI
L
N
P
M
Q
R
Vehicl
e0.3
10.6
31.2
5 2.5 50
50
100
150
200
Gro
wth
(% o
f veh
icle
)
NALM-6
3D
Compound 18 (µM)
2D*** *** *** ***
Vehicl
e0.3
10.6
31.2
5 2.5 50
50
100
150
OCI-LY18
Gro
wth
(% o
f veh
icle
)
3D
Compound 18 (µM)
2D*** ***
Vehicl
e0.3
10.6
31.2
5 2.5 50
50
100
150
NU-DUL-1
Gro
wth
(% o
f veh
icle
)
3D
Compound 18 (µM)
2D** * *** ***
IRE1a
XBP1s
GAPDH
NUDUL-1
OCI-LY18
NALM-6
O
Fig. S3
18
Figure S3 Biochemical characterization of compounds 18 and 16 and effect of 18
and 4µ8c on 3D versus 2D growth of B cell-derived cancer cells. (A) Biochemical
properties and kinase selectivity of compound 18, compound 16 and KIRA6. Kinase
inhibition was determined by competition for binding of a staurosporine-based probe to
the kinase pocket of a recombinant IRE1a protein comprising the kinase and
endoribunuclease moieties. RNase activity of the recombinant protein was measured by
cleavage of an XBP1s-based stem-loop structured RNA substrate as previously described
(6). XBP1s cell reporter assay was performed as previously described (3, 6) (see
Methods). Kinase-selectivity analysis against a panel of 220 kinases was performed at a
compound concentration of 1 µM by KinomeScanTM. (B) Schematic representation of the
kinase interactions of compound 18, compound 16 and KIRA6. Size and color of circles
are related to interaction strength, as indicated in the top right inset. (C) KMS-11 cells
were seeded on Matrigel to form multiple 3D spheroids, treated with serial dilutions of 18
or the JNK inhibitors JNK-IN-8 or SP600125 at the indicated concentrations, and then
analyzed for growth using an IncucyteTM S3 instrument. (D) Histogram of relative JNK2
mRNA expression in 1019 cancer cell lines in the cancer cell line encyclopedia (CCLE)
dataset (Broad Institute, Cambridge, MA, USA). (E) RPMI-8226 cells were incubated in
the absence or presence of serial dilutions of compound 18 for 8 hours and analyzed for
XBP1s mRNA levels by RT-qPCR (%XBP1s mRNA is the ratio of XBP1s
mRNA/(XBP1s mRNA+XBP1u mRNA). (F) KMS-11 cells were incubated for 8 hours
with Thapsigargin (Tg, 100 nM) in the absence or presence of serial dilutions of
compound 18 and analyzed by RT-qPCR for mRNA levels of XBP1s (blue) or the RIDD
target DGAT2 (red). Data in D and E are shown as the mean of triplicate determinations.
(G) Crystallographic statistics for the structure of compound 18 with IRE1a. (H-J) KMS-
11 IRE1a sh8-9 (H), OPM-2 IRE1a sh9 (I), and RPMI-8226 IRE1a shRNA7-5 cells (J)
were seeded either in the 2D or 3D setting, treated for 150 hours with either vehicle
(DMSO) or 4µ8c at the indicated concentrations, and then analyzed for cell growth using
an IncucyteTM instrument (H) or cell viability using CellTiter-Glo® 3D (I and J). (K-N)
Tumor cell proliferation trajectories determined by cell confluence using an IncucyteTM
instrument of cells shown in Fig. 3F (K and L), Fig. 3G (M) or Fig. 3H (N), seeded in
19
the 2D (D) or 3D (E-G) setting and treated with vehicle (DMSO) or compound 18 at the
indicated concentrations. (O-Q) Two diffuse large B-cell lymphoma (DLBCL) cell lines,
NU-DUL-1 (O) and OCI-LY18 (P), and the B cell precursor leukemia cell line Nalm-6
(Q) were seeded either in the 2D or 3D setting, treated for 150 hours with either vehicle
(DMSO) or compound 18 at the indicated concentrations, and analyzed for cell growth
using CellTiter-Glo® 3D. (R) IB analysis of IRE1a and XBP1s expression in the cell lines
depicted in O-Q. *p<0.05, **p≤0.01, ***p≤0.001.
20
0 5 10 15 20 250
1000
2000
Day
Tum
or v
olum
e (m
m3 )
Vehicle
0 4 8 120
2000
4000
Days
Tum
or v
olum
e (m
m3 )
Vehicle
0 4 8 120
2000
4000
Days
Tum
or v
olum
e (m
m3 )
Compound 18
0 5 10 15 20 250
1000
2000
Day
Tum
or v
olum
e (m
m3 )
Compound 18
A
Treatment group
N of animals day 0/
last day
AUC/ day % TGI mean
(range)
AUC/ day mean
(range)
1 Vehicle (5% sucrose) 15/14 0 (0, 0) 427
(314, 573)
2 Doxycyline(0.5 mg/mL, PO) 15/15 56
(31, 74)187
(126, 266)
3 Compound 18(30 mg/kg IP, BID) 15/14 51
(22, 70)209
(142, 290)
F
B
VentralDorsal
0 4 8 120
2000
4000
Days
Tum
or v
olum
e (m
m3 )
Dox
0 5 10 15 20 250
1000
2000
Day
Tum
or v
olum
e (m
m3 )
Dox
EDTreatment group
N of animals day 0/
last day
AUC/ day % TGI mean
(range)
AUC/ day mean
(range)
1 Vehicle (5% sucrose) 14/14 0 (0, 0) 583
(418,804)
2 Doxycyline(0.5 mg/mL, PO) 14/15 72
(50, 84)165
(103, 254)
3 Compound 18(30 mg/kg IP, QD) 14/14 70
(50, 84)172
(109, 225)
0 2 84 60.01
0.1
1
10
Hours
Tota
l blo
od c
onc
(µM) QD
BID
Compound 18 Compound 1830 mg/kg, QD
AUC (hr x µM) 9.3
Cmax (µM) 4.3
T1/2 (hr) 1.5
Vehicle Dox Compound 18
XBP1s
IRE1a
Actin
KMS-11 IRE1a sh8-9 xenografts
Vehicle
IRE1a
GAPDH
XBP1s
Dox Compound 18
OPM-2 IRE1a sh9 xenografts
C
Fig. S4
21
Fig. S4 Small-molecule inhibition of IRE1a kinase attenuates XBP1s production and
subcutaneous growth of human MM xenografts in mice. (A) C.B-17 SCID mice
bearing subcutaneous KMS-11 tumor xenografts (3 mice/ group) were treated
intraperitoneally (IP) with compound 18 (30 mg/kg) either as a single dose (QD), or
twice per day (BID) over 4 days. Plasma was collected at the indicated time after the last
dose and compound concentrations were determined by liquid chromatography and mass
spectrometry. The table summarizes pharmacokinetic parameters of compound 18 based
on single dose administration. (B) IB analysis to confirm depletion of XBP1s in
subcutaneous tumor xenografts sampled from individual mice depicted in Fig. 4A. (C)
Tumor growth trajectories of individual animals, corresponding to the mean tumor
volumes depicted in Fig. 4A. (D) IB analysis to confirm depletion of XBP1s in
subcutaneous tumor xenografts sampled from individual mice depicted in Fig. 4B. (E)
Tumor growth trajectories of individual animals, corresponding to the mean tumor
volumes depicted in Fig. 4B. (F) RPMI-8226 cells expressing plasmids encoding
mCherry and luciferase were intravenously injected via the tail vein. Multifocal
orthometastatic growth in the bone marrow with typical skeletal lesions in the skull
(white dashed box) and spine (yellow dashed box) was confirmed by live
bioluminescence (left-hand panels), post-mortal fluorescence imaging (middle panel), or
fluorescence co-registered with X-ray imaging (right-hand panel) within same animals.
22
A
Cohort Patient # Age, Sex Disease Type Disease State
BM MM (%) FISH/ QPCR Prior
Lines Prior Treatments Include
USA
576T1 45, F MM Diagnosis 40-50 Hyperdiplpoid13q-; IgH+ 0 None
1003 60, F MM Diagnosis 25-30 1q+,13q-, IgH+ 0 None
1229 52, M MM Diagnosis 40 N/A 0 None
576T3 47, F MM Relapsed 40 Hyperdiploid, 13q- 1 CTX, BTZ, DM; HDM-ASCT;
observation
1055 61, M MM Relapsed 10-15 t(X;4)+ 1 BTZ, DM
700T2 67, M MM Relapsed 30 t(11;14)+; 1q+ 2 CTX, BTZ, DM; HDM-ASCT; observation; LEN, DM
614T2 60, F MM Relapsed 5-10Hyperdiploid,
t(11;14)+, 13q-, 1q+
4
CTX, BTZ, DM; carfilzomib, LEN, DM; HDM-ASCT; BTZ, LEN, DM; elotuzumab, LEN, BTZ, DM; POM, DM
1070 65, F MM Relapsed 20 t(11;14)+, 17p- 4 CTX, BTZ, DM; carfilzomib, LEN, DM; POM, DM; DARA
1322 51, F MM Relapsed 30-40 1q+,13q- 4BTZ, LEN, DM; DARA, POM, DM; DARA, BTZ, POM, DM; DARA, carfilzomib, POM, DM, biaxin
EU
101711 56, FPrimary
Plasma Cell Leukemia
Diagnosis 83 Hyperdiplpoid 0 None
051719 66, F MM pleural effusion Relapsed 38
t(4;14)-; del17+CCND1-;ITGB7-;
FRZB-2 BTZ; LEN
051720 70, FSecondary Plasma Cell Leukemia
Relapsed 25 t(4;14)-; del17-CCND1+ 3 BTZ; LEN; POM
071739 62, M MM Relapsed 70 t(4;14)-; del17-FRZB+ 2 BTZ; LEN
MM49 66, MSecondary Plasma Cell Leukemia
Relapsed 44
t(4;14)-; t(11;14)-; del17+ITGB7+
4 BTZ; LEN; POM; bendamustine
B
Vehicl
e 1.9 3.8 7.5 15 300
50
100
150
200
Viab
ility
(% o
f veh
icle
) Compound 184µ8c
USA cohort
µM
Fig. S5
23
Fig. S5 Patient characteristics and comparison of compound 18 and 4µ8c activity.
(A) Demographic, cytogenetic and treatment characteristics of bone marrow or peripheral
blood samples donated by MM patient cohorts in the USA and EU as depicted in Fig. 5.
BTZ, bortezomib; CTX, cyclophosphamide; DM, dexamethasone; DARA, daratumumab;
HDM-ASCT, high-dose melphalan with autologous bone marrow transplantation; LEN,
lenalidomide; POM, pomalidomide. (B) Patient bone marrow aspirate (patient #1322),
cultured for 48 hours with either vehicle (DMSO), compound 18, or 4µ8c at the indicated
concentrations. Samples were then analyzed for viability by flow cytometry, with gating
on CD138+ or CD138— cells. Data represent mean ± SEM of triplicate determinations.
24
Vehicl
e0.0
30.0
90.2
70.8
12.4
3 7.30
50
100
150
Via
bilit
y (%
of v
ehic
le)
Compound 18 (µM)
Rat islets
Vehicl
e0.0
30.0
90.2
70.8
12.4
3 7.30
50
100
Sec
rete
d in
sulin
(f
Mol
/ hou
r/ m
icro
isle
t)
Rat islets
Compound 18 (µM)
A B
CTissues, microscopically examined Weighed
Adrenal glands NoBone marrow (sternum) NoBrain YesEyes and optic nerve NoHeart YesIntestine (cecum, colon, duodenum, ileum, jejunum, rectum)
No
Kidneys YesLiver YesLungs YesOvaries YesPancreas NoPituitary gland NoSalivary glands NoSpleen YesStomach No Thymus YesGross lesions as noted NoD
Subm
andi
bula
rgl
and
Subl
ingu
algl
and
Panc
reas
Kid
ney
Live
rD
uode
num
Vehicle Compound 18 Vehicle Compound 18
E
Vehicl
e
Compound 180
50
100
150
200
ALT
U/ L
Vehicl
e
Compound 180
200
400
600
AST
U/ L
Vehicl
e
Compound 180
10
20
30
40
BUN
mg/
dL
Vehicl
e
Compound 180.25
0.30
0.35
0.40
0.45
0.50
Creatinine
mg/
dL
Vehicl
e
Compound 180.0
0.1
0.2
0.3
0.4
0.5
Bilirubin
mg/
dL
Vehicl
e
Compound 18100
150
200
250
300
Glucose
mg/
dL
Fig. S6
25
Fig. S6 IRE1a kinase inhibition preserves normal tissue homeostasis and histology.
Rat pancreatic islets were isolated, dissociated into single cells, replated in microtiter
wells (1000 cells/drop), and allowed to form 3D microtissues of ~120 µm in diameter
over 7 days using inSpheroTM technology. Microtissues (n=5 per treatment) were then
incubated for 7 days in with vehicle (DMSO) or compound 18 at the indicated
concentrations, and then (A) analyzed for cell viability by CellTiter-Glo®; or (B)
challenged with glucose (16.7 mM) for 1 hour and analyzed for insulin secretion by
ELISA. (C) Summary table of mouse tissues examined (with or without weighing as
indicated) to analyze the impact of 18 on histology. (D) Representative images of tissues
stained with hematoxylin and eosin from mice treated with either vehicle (n=4) or 30
mg/kg compound 18 (n=4) IP, BID for 7 days. (E) NOD/SCID/IL2rγ-/- mice were
treated with vehicle (n=12) or 18 (30 mg/kg, n=13) IP, QD for 3 weeks. On day 21 blood
was drawn and processed routinely for clinical chemistry analysis of the indicated serum
makers.
26
Supplemental references 1. Mali P, et al. (2013) RNA-guided human genome engineering via Cas9. Science
339(6121):823-826. 2. Callow MG, et al. (2018) CRISPR whole-genome screening identifies new
necroptosis regulators and RIPK1 alternative splicing. Cell Death Dis 9(3):261. 3. Chang TK, et al. (2018) Coordination between Two Branches of the Unfolded
Protein Response Determines Apoptotic Cell Fate. Mol Cell 71(4):629-636 e625. 4. Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain
in electron microscopy. J Cell Biol 17:208-212. 5. Pinheiro J, Bornkamp B, Glimm E, & Bretz F (2014) Model-based dose finding
under model uncertainty using general parametric models. Stat Med 33(10):1646-1661.
6. Korennykh AV, et al. (2009) The unfolded protein response signals through high-order assembly of Ire1. Nature 457(7230):687-693.