a, Immunoprecipitation of HIF-1β from tumor lysates
of sensitive (XP373), intermediate (XP391), and resistant (XP506 and XP169)
tumors from mice treated with either vehicle (Veh) or PT2399. (Samples are
labeled with “V” for vehicle-treated or “P” for
PT2399-treated followed by the mouse identifier.) b, Proximity
ligation assay detecting either HIF-2α + HIF-1β or
HIF-1α + HIF-1β heterodimers from vehicle- or PT2399-treated
sensitive (XP374) or resistant (XP296) tumors and summary of results across
responsive and resistant tumorgrafts. (Images representative of quantitative
data shown in graph.) Summary includes analyses from 11 vehicle-treated tumors
and 11 PT2399-treated tumors (3 fields were analyzed for each sample) in 5
sensitive, 3 intermediate, and 3 resistant tumorgraft trials. Scale bars = 20
µM. c, qRT-PCR for the indicated HIF-2 target genes in
PT2399 sensitive, intermediate, and resistant tumors treated with vehicle
(blue), PT2399 (red), or sunitinib (green). HIF-1 target genes
CA9, PGK1, and LDHA
included as negative controls. Excepting PGK1 and
LDHA, samples were available for n = 58
vehicle-treated tumors (Sensitive: n = 11; Intermediate:
n = 21; Resistant: n = 26),
n = 62 PT2399-treated tumors (Sensitive: n
= 15; Intermediate: n = 21; Resistant: n =
26), and n = 52 sunitinib-treated tumors (Sensitive:
n = 10; Intermediate: n = 23; Resistant:
n = 19). PGK1 and LDHA
were available for 24 tumors for each treatment group (Sensitive:
n = 6; Intermediate: n = 8; Resistant:
n = 10). d, Circulating tumor-produced hVEGF
as well as mouse EPO levels in mice with sensitive, intermediate, and resistant
tumors treated with vehicle (blue), PT2399 (red), and sunitinib (green). ELISA
data was generated for 63 vehicle-treated tumors (Sensitive: n
= 21; Intermediate: n = 19; Resistant: n =
23), 74 PT2399-treated tumors (Sensitive: n = 27; Intermediate:
n = 21; Resistant: n = 26), and 61
sunitinib-treated tumors (Sensitive: n = 15; Intermediate:
n = 23; Resistant: n = 23).
e, Number of RNAs upregulated and downregulated genes by PT2399 in
sensitive and resistant tumors. f, Heatmap representation from
RNAseq analysis showing differentially-regulated genes by PT2399 in sensitive
compared to resistant tumors. Removal of an unclassified tumor (XP169) from the
resistant group, did not affect conclusions. g, RNAseq analyses
showing increased expression of selected genes by PT2399 in sensitive tumors.
b–d, g: Tests completed using a mixed model with
compound symmetrical covariance structure for mice in the same tumorgraft line
using vehicle as the reference group. qRT-PCR levels were log-transformed for
analysis; EPO and hVEGF levels were Box-Cox transformed; RNAseq levels were
log2-transformed; Raw values depicted in all graphs. All bar charts depict the
mean with the error bar representing s.e.m., while all boxplots have median
centre values. *, p < 0.05; **, p
< 0.01; ***, p < 0.001; and ****,
p < 0.0001. See Supplementary Fig. 1 for gel source
data.