Download Legends for Tables S1 to S3 and Figures S1 to S5

Supplementary Table and Figure Legends
Supplementary Table 1. qPCR primer sequences for a panel of BCL2 family members
(n=18) listed and referred to as Apopanel in the text. Sequences for a set of reference
house-keeping genes (TMEM55B, ACTB and VPS33B) used for normalizing RNA levels
are listed. Note: BCL2L1 gene encodes BCL-XL and BCL2L11 gene encodes BIM
Supplementary Table 2. Correlation of mRNA expression of BCL2 family members
listed in Table 1 with sensitivity to venetoclax (A) or navitoclax (B) in HMCLs. p-values
were determined by unpaired t-tests and sorted from lowest to highest. Ratio of BCL2 to
MCL1 or BCL2L1 RNA levels to sensitivity of venetoclax and navitoclax is included.
Note: BCL2L1 gene encodes BCL-XL and BCL2L11 gene encodes BIM
Supplementary Table 3. BCL2, BC2L1 and MCL1 mRNA expression in CD138
primary MM patient samples and sensitivity to venetoclax ex vivo. Expression of BCL2
(BCL-2), BCL2L1 (BCL-XL) and MCL1 (MCL-1) mRNA in purified CD138-positive
primary multiple myeloma patient samples were determined by TaqMan-based PCR as
described in Supplemental Materials and Methods. mRNA levels of each BCL2 gene
were normalized to the housekeeping gene, RPL37a, which served as an endogenous
control. Cell death of primary CD138+ myeloma plasma cells following treatment with
DMSO, as a vehicle control, or venetoclax at a dose range of 100 to 500 nM for 18 hours
was determined by flow cytometry using a combined loss of CD138 surface expression
and alternation of cellular morphology based on lower forward scatter (indicative of dead
cells). LD50 is the lethal dose of venetoclax that causes 50% cell death. Venetoclax
resistant cells were defined by a LD50 of > 500 nM.
Supplementary Figure 1. Disruption of BCL-2:BIM complexes in venetoclax-sensitive
HMCLs. KMS-28BM, KMS-12BM, KMS-11 and KMS-27 were treated with vehicle
(DMSO) control or venetoclax at the doses indicated for 24 hours. Following treatment,
HMCLs were harvested and processed for measuring BCL-2:BIM complexes as
described in Materials and Methods. Mean signal intensity is based on electrochemiluminescence.
Supplementary Figure 2. Immunoblots of BCL-2, BCL-XL and MCL-1 in HMCL
tumor xenografts. Untreated tumor xenografts of the models shown were analyzed for
BCL-2, BCL-XL and MCL-1 protein levels by Western blotting as described in Materials
and Methods. For MM.1S and NCI-H929 models one representative blot of a tumor
xenograft is shown for each protein while in the OPM-2 model examples from two
separate tumors are shown.
Supplementary Figure 3. Caspase dependent degradation of MCL-1 in NCI-H929
HMCL following treatment with bortezomib. A) NCI-H929 cells were maintained in
Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal
bovine serum. Cells were seeded at a density of 2 million cells per well in 6-well plates
and incubated overnight prior to treatment. Cells were treated with 10 nM bortezomib
alone or in combination 50 μM Z-VAD-FMK for 1, 4 and 8 hours. DMSO (Sigma) was
used as a vehicle control. Following treatments, cells were prepared for immunoblotting
against MCL-1, BCL-2 and Caspase 3 as described in Materials and Methods. Actin was
used as a loading control for total protein. B) Levels of MCL-1 relative to Actin in
immunoblots were quantified by densitometry using the LI-COR-Odyssey imaging
system.
Supplementary Figure 4. Conceptual workflow for establishing BCL-2 family IHC cutoffs for analysis of MM patient tumor samples. 1) and 2) HMCLs were tested in vitro for
sensitivity to venetoclax across a range of drug concentrations. Cell lines with a
venetoclax IC50 <1 M were considered “sensitive”; all other cell lines were considered
“resistant”. In the same cell lines, Western blotting and qPCR analyses assessed BCL-2
family protein and mRNA levels, respectively. Optimally predictive classifiers of drug
sensitivity were determined from BCL2 and BCL2L1 qPCR data. 2) and 3) In parallel,
formalin-fixed, paraffin-embedded (FFPE) multiple myeloma cell line samples were
stained for BCL-2 and BCL-XL; the IHC signal intensities that correlated with previously
identified qPCR BCL2 and BCL2L1 cutoffs for “sensitive” and “resistant” samples were
identified. These IHC intensity thresholds were then applied to patient FFPE bone
marrow biopsy samples to identify the patient population most likely to respond to
venetoclax.
Supplementary Figure 5. BCL-2 and BCL-XL IHC correlates with qPCR and
immunoblot analysis of HMCLs. A) Weak (1+), moderate (2+) and strong (3+) BCL-2
IHC signals are exemplified by KMS-34 (A), KMS-11 (B), and Amo-1 cell lines (C),
respectively. D) The correlations between IHC signal, qPCR and Western blot (WB)
quantification for 14 cell lines are shown. The column of BCL-2 IHC result values shows
IHC scores for replicate cell pellet samples in all but MOLP-8, for which only 1 sample
was available. Cell lines KMS-34, KMS-11 and Amo-1 are internal BCL-2 IHC staining
references for 1+, 2+ and 3+ signal, respectively. Pink and green highlighting indicates
cells lines that were below or above, respectively, the threshold of BCL-2 expression (dCt = 2.7) determined to most efficiently segregate sensitive from resistant lines (as
determined by in vitro IC50; see Supplemental Figure 3). “Low” (i.e., BCL-2 IHC signal
< 2+) and “High” (BCL-2 IHC signal ≥ 2+) indicate cell lines with BCL-2 IHC signal
below and above this threshold. E) Correlation between IHC score and Western blot
(WB) quantification of BCL-2 protein expression. Pink and green highlighted boxes
correspond to cell lines below and above, respectively, the threshold of BCL-2 expression
(-dCt = 2.7) determined to most efficiently segregate sensitive from resistant lines (as
determined by in vitro IC50; see Supplemental Figure 3). EJM and NCI-H929 are
imperfectly assigned to either group. F) Correlation between IHC score and qPCR
quantification of BCL2 expression. Pink and green highlighted boxes correspond to cell
lines below and above, respectively, the threshold of BCL2 expression (-dCt = 2.7)
determined to most efficiently segregate sensitive from resistant lines (as determined by
in vitro IC50; see Supplemental Figure 3). U266 and NCI-H929 are imperfectly assigned
to either group. E and F) Circled data points highlight expression levels of BCL-2 in cell
line samples used as reference standards for 1+, 2+ and 3+ IHC signal. B) BCL-XL IHC
correlates with qPCR and immunoblot analysis of HMCLs. Weak (1+), moderate (2+)
and strong (3+) BCL-2 IHC signals are exemplified by the KMS-31 (A), KMS-26 (B),
and MM.1S cell lines (C), respectively. D) The correlations between IHC signal, qPCR
and Western blot (WB) quantification for 14 cell lines are shown. The column of BCLXL IHC result values shows IHC scores for replicate cell pellet samples in all but MOLP8, for which only 1 sample was available. Cell lines KMS-11, KMS-26 and MM.1S are
internal BCL-XL IHC staining references for 1+, 2+ and 3+ signals, respectively. Pink
and green highlighting indicates cells lines that were above or below, respectively, the
threshold of BCL-XL expression (-dCt = 5) determined to most efficiently segregate
sensitive from resistant lines (as determined by in vitro IC50; see Supplemental Figure 3).
“Low” (i.e., BCL-XL IHC signal < 2+) and “High” (BCL-XL IHC signal ≥ 2+) indicate
cell lines with BCL-XL IHC signal below and above this threshold. E) Correlation
between IHC score and Western blot (WB) quantification of BCL-XL protein expression.
Pink and green highlighted boxes correspond to cell lines above or below, respectively,
the threshold of BCL-XL expression (-dCt = 5) determined to most efficiently segregate
sensitive from resistant lines (as determined by in vitro IC50; see Supplemental Figure 3).
KMS-34 is imperfectly assigned to either group. F) Correlation between IHC score and
qPCR quantification of BCL-XL expression. Pink and green highlighted boxes
correspond to cell lines below and above, respectively, the threshold of BCL-XL
expression (-dCt = 5). E and F) Circled data points highlight expression levels of BCL-2
in cell line samples used as reference standards for 1+, 2+ and 3+ IHC signal.