Multi-disciplinary efforts to evaluate the therapeutic potential of CDK11, a novel transcription associated cyclin dependent kinase. Christopher L. Carroll 1 , Jan Reiling 2 , Jeffrey J. Kovacs 2 , Michael Bradley 1 , Meng He 2 , Nadima Uprety 2 , Michael Peoples 2 , Frederick S. Robinson 2 , Alessandro Carugo 2 , Michael Conner 1 , Christian Rodriguez 1 , Jennifer Bardenhagen 1 , Vijayan Ramaswamy 1 , Paul G. Leonard 1 , Zhijun Kang 1 , Catalina Suarez 1 , Teresa L. Perry 2 , Jing Han 2 , Sonal Fnu 2 , Nakia D. Spencer 2 , Guang Gao 2 , Angela L. Harris 2 , Alan Xu 1 , Jennifer Linares 1 , Thomas M. Quill 1 , Phuong K. Nguyen 1 , Qi Wu 1 , Stephanie T. Schmidt 2 , Sahil Seth 2 , Christopher A. Bristow 2 , Norma E. Rogers 2 , Simon S. Yu 1 , Hannah E. Shepard 1 , Robert A. Mullinax 2 , Sabina A. Lorca 2 , Kang Le 1 , Reagan Lucas 1 , Sweta Mahaeshwari 1 , Sarah Joseph 3 , Ningping Feng 2 , Yongying Jiang 1 ,Joe Marszalek 2 , Michael J. Soth 1 , Jason B. Cross 1 , Mary K. Geck Do 1 , M. Emilia Di Francesco 1 , Timothy P. Heffernan 2 , Philip Jones 1 1 IACS (Institute for Applied Cancer Science); 2 TRACTION (Translational Research to AdvanCe Therapeutics and Innovation in Oncology), University of Texas MD Anderson Cancer Center, Houston, Texas 77054, United States; 3 Department of Genomic Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, United States
Identification of CDK11 as a novel therapeutic target We previously employed our in vivo target discovery platform, PILOT (Patient-based In vivo Lethality to Optimize Treatment), to identify new vulnerabilities that can be exploited therapeutically to optimize cancer treatment. Leveraging the PILOT platform, cyclin dependent kinase 11 (CDK11) was identified as a top-scoring hit from an in vivo functional genomics screen across multiple patient-derived PDAC xenograft models.
Kinase activity is critical to rescue CDK11 knockdown in cells Overexpression of CDK11 wild type (WT) but not kinase defective mutants (562 and 580) rescue the growth inhibitory effect of CDK11 knockdown in PDAC cell lines.
Profiles of in vivo tool compounds
Disconnect with pharmacological inhibition Pharmacological inhibition of CDK11 induced a potent anti- proliferative response across a panel of PDAC cell lines, in contrast to the differential response observed with genetic disruption. (IACS- 074418 is closely related to IACS-074527 with a comparable profile)
Selective alterations of replication-dependent histone gene expression
Potent and selective tool compounds with suitable pharmacokinetic properties for oral or intraperitoneal (IP) dosing were identified. Compounds are low nanomolar in biochemical and cellular target engagement assays and induce a potent anti-proliferative response in PATC53 cells. Both compounds exhibit selectivity over the CDK family and the broader kinome (>100x over kinome panel). Series 2 compound, IACS-054647 , exhibits an excellent in vitro and in vivo pharmacokinetic profile, suitable for oral dosing in mouse models. Series 3 compound, IACS-074527 , suffers from poor permeability and low solubility, limiting exposures with oral dosing. However, low metabolic clearance allows for excellent exposure with IP dosing.
No differential response observed in PDAC panel
100
100
80
PATC53
PILOT in vivo functional genomics screen
80
PATC53
PATC153
PATC153
60
60
PATC124
PATC124
40
40
PATC148
PATC148
20
20
Kinase-dead
Kinase-dead
Pleiotropic functions of CDK11
0
0
0.0001
0.01
1
0.0001
0.01
1
IACS-74418 [ M]
IACS-54647 [ M]
Minimal tumor growth inhibition observed at tolerated doses in PATC53 mouse efficacy models
Mechanism of action studies uncovered pleiotropic functions of CDK11, including time dependent impact on RNA metabolism, altered cell cycle function, and selective alteration of replication-dependent histone gene expression.
Gene modulation in PATC53 •
22 genes with 2 fold decrease 44 genes with 1.5 fold decrease
•
IACS-054647 mouse efficacy/tolerability study
Vehicle IACS-054647, 20 mpk PO, QD* IACS-054647, 10 mpk PO, QD
1500
Differential response across PDAC cell lines Genetic disruption of CDK11 expression induced a differential response across a panel of PDAC cell lines in vitro. Knockdown of CDK11 inhibited the growth established xenografts in vivo.
Identification of small molecule inhibitors
CDK11 knockdown alters splicing patterns
Body weight change
1000
IACS-054647 (Series 2)
IACS-074527 (Series 3)
10
0
Given the biological findings, CDK11 represented an opportunity to develop a first-in-class small molecule inhibitor. A screening funnel was established to enable the development of high quality tool compounds. Two chemically distinct leads were identified from a high-throughput screening campaign. Weekly profiling of analogs in activity assays and in vitro pharmacokinetic assays allowed for rapid optimization. Select compounds were then profiled in the PATC53 cellular phenotypic assay, kinase selectivity panels and mouse pharmacokinetic studies to identify in vivo tool compounds.
-10
500
CDK11A/CycD3 IC 50 ( M)
0.006
0.001
-20
-30
0
5
10
15
20
CDK11A/CycL2 Cell IC 50 ( M) PATC53 Phenotypic EC 50 ( M) CDK9/T1 2 mM ATP IC 50 ( M) CL int mouse LM (mL/min/kg)
0.013
0.018
Treatment (Days)
0
0.303
0.320
R
0
5
10
15
20
NR
Treatment (Days)
3.6
2.5
*20 mpk dose not tolerated (4 mice required dosing holidays)
53
15
IACS-074527 mouse efficacy/tolerability study
-6 cm/s)
MDCK P app A-B (x10
19
<0.4
Vehicle, IP QD IACS-074527, 40 mpk IP, QD* IACS-074527, 5 mpk IP, QD
MDR1 ER ((B-A)/(A-B))
1.9
>90
1500
Solubility pH = 7.4 ( M)
62
16
Body weight change
CL p , IV 1 mg/kg (mL/min/kg) V ss , IV 1 mg/kg (L/kg) T 1/2 , IV 1 mg/kg (h) C max , 10 mg/kg ( M) AUC last ,10 mg/kg (h* M)
26.6
22.2
10
1000
PATC124
PATC69
Vehicle, IP, QD IACS-074527, 4 IACS-074527, 5
0
8.3
13.2
1.5
1.5
1.5
1.5
-10
Drug discovery screening funnel
5.8
10.3
500
1.0
1.0
1.0
-20
1.0
2.81 ± 0.773 †
4.07 ± 1.21 ‡
Biochemical Assay CDK11A/CyclinD3 RDA
Selectivity Assay CDK9/CyclinT1 LANCE
-30
Significant increase of differential splicing events (q < 0.05) in PATC53
0.5
0.5
0.5
0.5
0
5
10
15
20
0
†
8.13 ‡
25.8
Treatment (Days)
0
5
10
15
20
Differential regulation of cell cycle
0.0
0.0
0.0
0.0
135 † 60.5 ‡ † Oral dosing; ‡ Intraperitoneal dosing
Treatment (days)
F, 10 mg/kg (%)
IC 50 ≤0.1 M
*40 mpk dose not tolerated (required dosing holidays for days 8 – 15)
PATC53 sh3+rescue
PATC53 shP2
PATC53 shLUC
PATC53 sh3
PATC53 shP2+rescue
Kinase selectivity panels Compounds were screened in a select panel of kinases, including the entire CDK family, based on full kinome scans with representative compounds in each series.
Cellular Target Engagement Assay CDK11A/CyclinL2 NanoBRET (HEK293)
No Dox
Dox
No Dox
Dox
No Dox
Dox
No Dox
Dox
In Vitro Pharmacokinetics LM’s, PPB, Sol.
Conclusions • CDK11 was identified from an in vivo functional genomics screen as a novel therapeutic target in PCAD patients. • Pharmacological inhibition with potent and selective tool compounds revealed a disconnect with genetic validation data. • Minimal tumor growth inhibition was observed in mouse efficacy studies as exposures were limited due to toxicity. • Taken together, our multidisciplinary approach to evaluate genetic and pharmacological inhibition of CDK11 suggests that while there is a dependency of a subset of tumor cells on CDK11, systemic inhibition of CDK11 in a whole organism is not tolerated, thereby limiting the therapeutic potential.
G1
R
PATC53
S
PATC124
1000 1200 1400 1600 1800
G2
900
shLUC off dox shLUC ON dox
shLuc Off DOX
IACS-054647
IACS-074527
IC 50 ≤0.2 M
37.2 % G1 43.4 % S 16.7 %G2
39.8 % G1 29.7 % S 28.8 %G2
36.2 % G1 45.5 % S 16.3 %G2
800
34.8 % G1 36.4 % S 25.7 %G2
42.6 % G1 36.4 % S 19.9 %G2
shLuc On DOX
700
CDK13/CycK CDK9/CycT1
CDK9/CycT1 151x CDK7/CycH/MAT1 902x CDK13/CycK 1486x CDK12/CycK 1653x CDK10/CycQ 2338x All other CDKs >5000 HIPK4 317x MEK2 1319x
272x
Cellular Phenotypic Assay CyQuant (PATC53) IC 50 ≤0.5 M, >100x selective %F >50
sh3 off dox sh3 on DOX
Selectivity Panels CDK family/Kinome
600
sh3 Off DOX
PATC124 shLUC
PATC124 shP2
PATC124 sh3
332x CDK10/CycQ 398x CDK12/CycK 414x CDK16/CycY 506x All other CDKs >800x TAOK2 284x ERK7 482x
500
sh3 On DOX
200 400 600 800
400
G1
300
Mouse Pharmacokinetics
NR
200
S
100
0 3 6 9 12151821242730 0
G2
0 3 6 9 1215182124273033363942 0
41.2 % G1 45.9 % S 11.0 %G2
46.0 % G1 38.4 % S 13.9 %G2
47.3 % G1 36.0 % S 14.0 %G2
In Vivo tool compounds
Days on Treatment
Days on Treatment
Fold selectivity based on IC 50 values
Fold selectivity based on IC
50 values
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