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: aa@gene.com 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.