A s P C 1 J V E - 1 0 9 C A P A N 2 S N U - 1 4 1 1 H P A ... · Spranger et al; Nature Reviews...
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#3874 Figure 1: Upstream alterations trigger aberrant Wnt ligand- dependent signalling RNF43/ZNRF3 keep surface Fzd in check, allowing the destruction complex to phosphorylate and degrade β-catenin - Wnt pathway ‘OFF’. Loss-of-function (LOF) RNF43/ZNRF3 mutations (1), or high RSPO expression through gene fusion (2), ultimately leads to accumulation of β-catenin - Wnt pathway ‘ON’. Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancer Simon Woodcock , Inder Bhamra, Clifford Jones, Alicia Edmenson Cook, Catherine Eagle and Caroline Phillips Redx Pharma, Block 33S, Mereside, Alderley Park, Cheshire, SK10 4TG, UK; e: [email protected]; t: +44(0)1625 469937; www.redxpharma.com Introduction References Signalling through the Wnt pathway is highly regulated at the level of ligand (Wnt), receptor (Fzd/LRP) and downstream components (e.g. destruction complex – APC/Axin/GSK3β). Post- translational modification of Wnt ligands via porcupine (PORCN; a membrane bound O- acyltransferase) is essential for secretion of active Wnt 1 . Activity of RNF43/ZNRF3 (E3-ubiquitin ligases) results in ubiquitination and membrane clearance of Fzd, whilst RNF43/ZNRF3 levels are kept in check via LGR and secreted RPSO ligands 2 (Fig. 1). The potent and selective porcupine (PORCN) inhibitor RXC004 is being investigated in a Phase 1 clinical trial (NCT03447470) 3 , and has the potential to treat tumours dependent on Wnt-ligand. Upstream Wnt pathway aberrations, including RNF43/ZNRF3 mutations and RSPO-fusions, result in high levels of surface Fzd receptors and increased Wnt-ligand dependent signalling 4 (Fig. 1). These aberrations are implicated in pancreatic, gastric and colorectal cancer (CRC). Dysregulated Wnt signalling initiates oncogenic pathways involved in tumour initiation, growth and metastasis 5 , and is more recently linked to tumour immune evasion 6,7 (see also abstract #506). 1. Biechele et al; Dev Biol, 2011; 355(2):275-285. 2. Zhan et al; Oncogene, 2017; 36:1461-1473. 3. https://clinicaltrials.gov /. 4. Madan et al; Oncogene, 2016; 35(17):2197-2207. 5. Anastas et al; Nat. Rev. Cancer, 2013, 13 (1): 11-26. 6. Wang et al; TIPs, 2018, 39(7):648. 7. Spranger et al; Nature Reviews 18:139 2018. Summary Results Anti-proliferative effects of RXC004 in genetically-defined tumour cell lines RXC004 efficacy and pathway inhibition translates in vivo Figure 2: RXC004 anti-proliferation potency in genetically-defined pancreatic and CRC cell lines. (A) RXC004 was evaluated across a panel of 7 genetically-defined tumour cell lines. (B) Indicated cell lines were treated with a dose response of RXC004 for 5 days, proliferation was measured using an ATP-lite assay. N≥3 throughout. Cell lines harbouring RNF43/ZNRF3 mutations or RSPO-fusions are sensitive to RXC004 as predicted, with anti-proliferative effects ranging from 0.3nM to 7nM. Figure 5. RXC004 demonstrates in vivo efficacy with associated pharmacodynamic (PD) changes in genetically-defined human xenograft models Relative tumour volumes (A, C), end of study tumour weights (B; mg), end of study relative gene expression of Axin2, cMyc, RNF43, MMP7, CD44, MUC4 and MUC5AC (D-F), and histology (G, H) were analysed. HPAF-II (A, D, G; 5x10 6 cells; athymic nude mice), AsPC1 (B, E; 3x10 6 cells; athymic nude mice), and SNU-1411 (C, F, H; 1x10 7 cells; NOC-SCID mice) were implanted subcutaneously. Treatment was initiated once tumour volumes reached ~100-150mm 3 (A- H). Dosing was 1.5mg/kg BID RXC004 for 7-13 days then QD for the remainder of study (A-C). Tumour RNA was isolated for RT-qPCR expression analysis of the indicated genes, relative to appropriate housekeepers, at 12h post final dose (D-F). End of study HPAF-II (G) and SNU-1411 (H) tumours were formalin- fixed paraffin-embedded (FFPE) and sections stained with haematoxylin and eosin (H&E), anti-Ki67 (proliferation marker) or combined Alcian blue-PAS (Ab_PAS; Mucin differentiation marker) as indicated. Mann-Whitney U (A-C) or unpaired t-test (D-F) p values. Figure 3: Effects of RXC004 treatment on the cell cycle Cells were treated with RXC004 (100nM) or vehicle (0.1% DMSO) for 72h. Cell cycle profiles were determined using propidium iodide (A, C) and phospho-Ser10-Histone H3 (mitotic marker; B, D) staining by flow cytometry. Data are N≥3 except for HCT116 and AsPC1 (N=1). Representative flow plots for cell cycle (C) and phospho-Histone H3 (D) in HPAF-II cells treated as indicated. T-test p values. Figure 4. RXC004 regulates multiple downstream Wnt pathway effectors in genetically-defined pancreatic and CRC tumour cell lines (A) A custom TaqMan qPCR gene array was designed based on literature evidence for genes modulated by the Wnt pathway. These custom 96-well arrays were used to screen the expression of 14 potential RXC004 effector genes (including the 9 genes indicated in A), across 7 genetically defined tumours lines (Fig. 2). Cells were treated with RXC004 (10nM) or vehicle (0.1% DMSO) for 3 days, total RNA was extracted and gene expression assessed relative to appropriate housekeepers. Heatmap (A) indicates the relative increase (green) or decrease (red) in gene expression induced by RXC004 treatment when compared to DMSO control. (B) Individual TaqMan qPCR assays for the 8 target genes indicated were confirmed separately in the specified cells lines. Cells were treated with RXC004 (10 or 1000nM) or vehicle (0.1% DMSO) for 3 days, total RNA was extracted and gene expression assessed relative to appropriate housekeepers. (C) Indicated cell lines were treated with a dose response of RXC004 for 3 days, total RNA was extracted and gene expression assessed relative to appropriate housekeepers. IC50 values were determined for the down-regulation of Axin2 and cMyc gene expression, N≥3. RXC004 arrests at the G1/S and G2/M cell cycle checkpoints Efficacy and sustained Wnt pathway inhibition by low and scheduled RXC004 Figure 6. RXC004 displays sustained in vivo PD effects and efficacy at low doses in the human SNU-1411 colorectal xenograft model Pharmacokinetic (PK) analysis of RXC004 (A, B), pharmacodynamic (PD) analysis of gene expression (C, D), tumour volume measurements (E), and histology (F). SNU-1411 (1x10 7 cells; NOC-SCID mice) were implanted subcutaneously, treatment was initiated at 200mm 3 (A-D) or 125mm 3 (E). Dosing of RXC004 was 1.5mg/kg (A, C) or 5mg/kg (B, D) continuous for 7 days, or as indicated for 19 days (E; 5/2 – 5 days on, 2 days off dosing). Tumour RNA was isolated for RT-qPCR expression analysis of the indicated genes at the specified timepoints post final dose (C, D). End of study tumours from (A-D; 7 days dosing) were FFPE and sections stained with H&E, anti-Ki67 or Ab_PAS as indicated (F). N3 per timepoint (A-D), N9 per group (E). Ordinary one-way ANOVA p values (A-E). RXC004 inhibits tumour proliferation and increases differentiation A B C D A B In vitro pathway inhibition by RXC004 in genetically-defined tumour cell lines AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO 32 16 8 4 2 2 4 8 16 32 UP DOWN AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO 32 16 8 4 2 2 4 8 16 32 UP DOWN AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO 32 16 8 4 2 2 4 8 16 32 UP DOWN 64 256 AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO UP DOWN 32 16 8 4 2 2 4 8 16 32 64 256 AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO 32 16 8 4 2 2 4 8 16 32 UP DOWN AXIN2 MYC CD44 MMP7 MUC2 MUC4 MUC5AC RNF43 Fold-change vs DMSO UP DOWN 32 16 8 4 2 2 4 8 16 32 64 256 WiDr JVE-109 SNU-1411 AsPC1 CAPAN2 HPAF-II β-Catenin Wnt Tumour Cells Tumour cell proliferation Regulatory T cells Treg activation Wnt MDSC Recruitment Of MDSC Rho JNK Ca 2+ β-catenin dependent pathway β-catenin independent pathway Tumour Cell RXC004 RXC004 Dendritic Cell CD8 T Cell Expansion and infiltration of T-cells Recruitment. Induction of Regulatory DC CXCL9 Tumour cell differentiation Direct tumour targeting – genetically defined Immuno-modulatory Gene expression changes (Log2 values) from DMSO control induced by RXC004 Undetected Undetected A B C e D 0 50 100 150 Axin2 Relative Axin2 mRNA levels *** e D 0 50 100 150 cMyc Relative cMyc mRNA levels * e D 0 50 100 150 RNF43 Relative RNF43 mRNA levels * e D 0 50 100 150 CD44 Relative CD44 mRNA levels * 0 200 400 600 MUC5AC Relative MUC5AC mRNA levels *** e D 0 500 1000 1500 MUC4 Relative MUC4 mRNA levels * e D 0 50 100 150 Axin2 Relative Axin2 mRNA levels **** e D 0 50 100 150 cMyc Relative cMyc mRNA levels ** e D 0 50 100 150 RNF43 Relative RNF43 mRNA levels ** e D 0 50 100 150 MMP7 Relative MMP7 mRNA levels * e D 0 2000 4000 6000 8000 MUC4 Relative MUC4 mRNA levels * 0 100 200 300 400 500 MUC5AC Relative MUC5AC mRNA levels * e D 0 50 100 150 Axin2 Relative Axin2 mRNA levels *** e D 0 50 100 150 cMyc Relative cMyc mRNA levels ** e D 0 50 100 150 RNF43 Relative RNF43 mRNA levels ** e D 0 50 100 150 MMP7 Relative MMP7 mRNA levels *** 0 100 200 300 400 MUC5AC Relative MUC5AC mRNA levels * e D 0 100 200 300 400 MUC4 Relative MUC4 mRNA levels ** HPAF-II 100 1000 Wet Tumour Weight (mg) * Vehicle BID RXC004 1.5mg/kg BID 300 AsPC-1 10 Relative Tumour Volume *** 3 30 Vehicle BID RXC004 1.5mg/kg BID SNU-1411 Relative Tumour Volume ✱ Vehicle BID RXC004 1.5mg/kg BID 3 5 1 2 A HPAF-II AsPC-1 SNU-1411 B C D E F H & E Ki67 - proliferation Ab_PAS - Mucins Vehicle RXC004 H & E Ki67 - proliferation Ab_PAS - Mucins Vehicle RXC004 G H 2hr 6hr 12hr 24hr 0 500 1000 1500 2000 2500 MUC4 expression MUC4 mRNA levels relative to vehicle (%) * 2hr 6hr 12hr 24hr 0 200 400 600 800 MUC5AC expression MUC5AC mRNA levels relative to vehicle (%) * * **** 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 AXIN2 expression Axin2 mRNA levels relative to vehicle (%) ** ** *** * 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 cMYC expression cMYC mRNA levels relative to vehicle (%) * * * 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 RNF43 expression RNF43 mRNA levels relative to vehicle (%) ** * * 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 CD44 expression CD44 mRNA levels relative to Vehicle (%) * ** ** 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 AXIN2 expression Axin2 mRNA levels relative to vehicle (%) ** ** ** ** 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 cMYC expression cMYC mRNA levels relative to vehicle (%) *** *** *** *** 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 RNF43 expression RNF43 mRNA levels relative to vehicle (%) ** ** * * 2hr 6hr 12hr 24hr 0 20 40 60 80 100 120 CD44 expression CD44 mRNA levels relative to Vehicle (%) * *** ** ** 2hr 6hr 12hr 24hr 0 500 1000 1500 2000 2500 MUC4 expression MUC4 mRNA levels relative to vehicle (%) **** ** * *** 2hr 6hr 12hr 24hr 0 200 400 600 800 MUC5AC expression MUC5AC mRNA levels relative to vehicle (%) * **** ** ** A B C D E F H & E Ki67 - proliferation Ab_PAS - Mucins Vehicle QD RXC004 1.5mg/kg QD RXC004 5mg/kg QD Vehicle QD RXC004 1.5mg/kg QD RXC004 5mg/kg QD RXC004 1.5mg/kg BID RXC004 1.5mg/kg BID 5/2 0 500 1000 1500 2000 Day 19: SNU-1411 tumours Tumour Volume (mm 3 ) * ** ** ** Data represent Mean ±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001
Transcript of A s P C 1 J V E - 1 0 9 C A P A N 2 S N U - 1 4 1 1 H P A ... · Spranger et al; Nature Reviews...
#3874
Figure 1: Upstream alterationstrigger aberrant Wnt ligand-dependent signallingRNF43/ZNRF3 keep surface Fzd incheck, allowing the destructioncomplex to phosphorylate anddegrade β-catenin - Wnt pathway‘OFF’. Loss-of-function (LOF)RNF43/ZNRF3 mutations (1), orhigh RSPO expression throughgene fusion (2), ultimately leadsto accumulation of β-catenin -Wnt pathway ‘ON’.
Efficacy of the Wnt/Beta-Catenin pathway inhibitor RXC004 in genetically-defined models of cancerSimon Woodcock, Inder Bhamra, Clifford Jones, Alicia Edmenson Cook, Catherine Eagle and Caroline Phillips
Redx Pharma, Block 33S, Mereside, Alderley Park, Cheshire, SK10 4TG, UK; e: [email protected]; t: +44(0)1625 469937; www.redxpharma.com
Introduction
References
Signalling through the Wnt pathway is highly regulated at the level of ligand (Wnt), receptor(Fzd/LRP) and downstream components (e.g. destruction complex – APC/Axin/GSK3β). Post-translational modification of Wnt ligands via porcupine (PORCN; a membrane bound O-acyltransferase) is essential for secretion of active Wnt1. Activity of RNF43/ZNRF3 (E3-ubiquitinligases) results in ubiquitination and membrane clearance of Fzd, whilst RNF43/ZNRF3 levels arekept in check via LGR and secreted RPSO ligands2 (Fig. 1).
The potent and selective porcupine (PORCN) inhibitor RXC004 is being investigated in a Phase 1clinical trial (NCT03447470)3, and has the potential to treat tumours dependent on Wnt-ligand.Upstream Wnt pathway aberrations, including RNF43/ZNRF3 mutations and RSPO-fusions, resultin high levels of surface Fzd receptors and increased Wnt-ligand dependent signalling4 (Fig. 1).These aberrations are implicated in pancreatic, gastric and colorectal cancer (CRC). DysregulatedWnt signalling initiates oncogenic pathways involved in tumour initiation, growth andmetastasis5, and is more recently linked to tumour immune evasion6,7 (see also abstract #506).
1. Biechele et al; Dev Biol, 2011; 355(2):275-285. 2. Zhan et al; Oncogene, 2017; 36:1461-1473. 3. https://clinicaltrials.gov/. 4. Madan et al; Oncogene, 2016; 35(17):2197-2207. 5. Anastas et al; Nat. Rev. Cancer, 2013, 13 (1): 11-26. 6. Wang et al; TIPs, 2018, 39(7):648. 7. Spranger et al; Nature Reviews 18:139 2018.
Summary
ResultsAnti-proliferative effects of RXC004 in genetically-defined tumour cell lines RXC004 efficacy and pathway inhibition translates in vivo
Figure 2: RXC004 anti-proliferation potency in genetically-defined pancreatic and CRC cell lines.(A) RXC004 was evaluated across a panel of 7 genetically-defined tumour cell lines. (B) Indicated cell lines were treated with a dose response of RXC004for 5 days, proliferation was measured using an ATP-lite assay. N≥3 throughout. Cell lines harbouring RNF43/ZNRF3 mutations or RSPO-fusions aresensitive to RXC004 as predicted, with anti-proliferative effects ranging from 0.3nM to 7nM.
Figure 5. RXC004 demonstrates in vivo efficacy with associated pharmacodynamic (PD) changes in genetically-defined human xenograft modelsRelative tumour volumes (A, C), end of study tumour weights (B; mg), end of study relative gene expression of Axin2, cMyc, RNF43, MMP7, CD44, MUC4and MUC5AC (D-F), and histology (G, H) were analysed. HPAF-II (A, D, G; 5x106 cells; athymic nude mice), AsPC1 (B, E; 3x106 cells; athymic nude mice), andSNU-1411 (C, F, H; 1x107 cells; NOC-SCID mice) were implanted subcutaneously. Treatment was initiated once tumour volumes reached ~100-150mm3 (A-H). Dosing was 1.5mg/kg BID RXC004 for 7-13 days then QD for the remainder of study (A-C). Tumour RNA was isolated for RT-qPCR expression analysis ofthe indicated genes, relative to appropriate housekeepers, at 12h post final dose (D-F). End of study HPAF-II (G) and SNU-1411 (H) tumours were formalin-fixed paraffin-embedded (FFPE) and sections stained with haematoxylin and eosin (H&E), anti-Ki67 (proliferation marker) or combined Alcian blue-PAS(Ab_PAS; Mucin differentiation marker) as indicated. Mann-Whitney U (A-C) or unpaired t-test (D-F) p values.
Figure 3: Effects of RXC004 treatment on the cell cycleCells were treated with RXC004 (100nM) or vehicle (0.1% DMSO) for 72h. Cell cycle profiles weredetermined using propidium iodide (A, C) and phospho-Ser10-Histone H3 (mitotic marker; B, D)staining by flow cytometry. Data are N≥3 except for HCT116 and AsPC1 (N=1). Representative flowplots for cell cycle (C) and phospho-Histone H3 (D) in HPAF-II cells treated as indicated. T-test p values.
Figure 4. RXC004 regulates multiple downstream Wnt pathway effectors in genetically-defined pancreatic and CRC tumour cell lines(A) A custom TaqMan qPCR gene array was designed based on literature evidence for genes modulated by the Wnt pathway. These custom 96-well arrayswere used to screen the expression of 14 potential RXC004 effector genes (including the 9 genes indicated in A), across 7 genetically defined tumours lines(Fig. 2). Cells were treated with RXC004 (10nM) or vehicle (0.1% DMSO) for 3 days, total RNA was extracted and gene expression assessed relative toappropriate housekeepers. Heatmap (A) indicates the relative increase (green) or decrease (red) in gene expression induced by RXC004 treatment whencompared to DMSO control. (B) Individual TaqMan qPCR assays for the 8 target genes indicated were confirmed separately in the specified cells lines. Cellswere treated with RXC004 (10 or 1000nM) or vehicle (0.1% DMSO) for 3 days, total RNA was extracted and gene expression assessed relative toappropriate housekeepers. (C) Indicated cell lines were treated with a dose response of RXC004 for 3 days, total RNA was extracted and gene expressionassessed relative to appropriate housekeepers. IC50 values were determined for the down-regulation of Axin2 and cMyc gene expression, N≥3.
RXC004 arrests at the G1/S and G2/M cell cycle checkpoints
Efficacy and sustained Wnt pathway inhibition by low and scheduled RXC004
Figure 6. RXC004 displays sustained in vivo PD effects and efficacy atlow doses in the human SNU-1411 colorectal xenograft modelPharmacokinetic (PK) analysis of RXC004 (A, B), pharmacodynamic (PD)analysis of gene expression (C, D), tumour volume measurements (E), andhistology (F). SNU-1411 (1x107 cells; NOC-SCID mice) were implantedsubcutaneously, treatment was initiated at 200mm3 (A-D) or 125mm3 (E).Dosing of RXC004 was 1.5mg/kg (A, C) or 5mg/kg (B, D) continuous for 7days, or as indicated for 19 days (E; 5/2 – 5 days on, 2 days off dosing).Tumour RNA was isolated for RT-qPCR expression analysis of theindicated genes at the specified timepoints post final dose (C, D). End ofstudy tumours from (A-D; 7 days dosing) were FFPE and sections stainedwith H&E, anti-Ki67 or Ab_PAS as indicated (F). N3 per timepoint (A-D),N9 per group (E). Ordinary one-way ANOVA p values (A-E).
RXC004 inhibits tumour proliferation and increases differentiation
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44 m
RN
A levels
rela
tive
to
Ve
hic
le (
%)
*****
2hr 6hr 12hr 24hr0
20
40
60
80
100
120AXIN2 expression
Axin
2 m
RN
A levels
rela
tiv
e t
o v
eh
icle
(%
)
**** ** **
2hr 6hr 12hr 24hr0
20
40
60
80
100
120cMYC expression
cM
YC
mR
NA
levels
rela
tiv
e t
o v
eh
icle
(%
)
*** ***
******
2hr 6hr 12hr 24hr0
20
40
60
80
100
120RNF43 expression
RN
F43 m
RN
A levels
rela
tiv
e t
o v
eh
icle
(%
)
** ****
2hr 6hr 12hr 24hr0
20
40
60
80
100
120CD44 expression
CD
44 m
RN
A levels
rela
tive
to
Ve
hic
le (
%)
******
**
2hr 6hr 12hr 24hr0
500
1000
1500
2000
2500 MUC4 expression
MU
C4 m
RN
A levels
rela
tiv
e t
o v
eh
icle
(%
)
****
** *
***
2hr 6hr 12hr 24hr0
200
400
600
800 MUC5AC expression
MU
C5A
C m
RN
A levels
rela
tiv
e t
o v
eh
icle
(%
)
*
****
** **
A B
C D
E
F H & E Ki67 - proliferation Ab_PAS - Mucins
Ve
hic
le Q
D
RX
C0
04
1
.5m
g/k
g Q
DR
XC
00
4
5m
g/k
g Q
D
Vehicle QD
RXC004 1.5mg/kg QD
RXC004 5mg/kg QD
RXC004 1.5mg/kg BID
RXC004 1.5mg/kg BID 5/2
0
500
1000
1500
2000 Day 19: SNU-1411 tumours
Tu
mo
ur
Vo
lum
e (
mm
3)
* ** ** **
Data represent Mean ±SEM. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001
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