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Regulation of Bone by Wnt Pathway

Anna Georgieva

Ruben Bibas, Gabriel Helmlinger, Brian StollModeling and Simulation, Novartis Pharma

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Outline

Introduction: modeling platformsBiology

Bone Wnt signaling pathwayRegulation

ModelingWntBoneRegulation

Example: PK/PD of a therapeutic agentConclusions

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Modeling ofbiological and (patho-)physiological processes

SystemsBiology

Pharmacokinetics Pharmacodynamics

Cellular-levelTissue-level VEGF Molecular-level

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Modeling platforms

Model biology system of interestSystematic incorporation of data

may serve as institutional memoryguide new experiments

Ability to address cross-program comparisons Allow for prioritization of projects within same programExtraction of PD model sub-structures as neededAbility to conduct in silico experiments when direct experimental measurements are not possibleTeam effort is essential!

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Bone cell biology

Bone mass: balance between osteoclast (OC) and osteoblast (OB) activity

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Bone biology: differentiation process for OC, OB and CC

Osteoblasts

Early Chondrocyte

Runx2

lhh

OsxB-catenin/TCF/LEF

Sox9 Sox5L Sox6 B-catenin /TCF/LEF

BMP�s FGF�s Wnt�s

BMP�s FGF�s

lhh Wnt�s PRHrP�sColumnar Chondrocyte

Hypertrophic Chondrocyte

BMP�s

Runx2/3

Dix5/6

Osteoblast Progenitor

Osteocytes

Mesenchymal Cells

Twist1,2M-CSF RANKL

Marrow Precursors

PU,1 Fos/Fra1NFATc1NFkBMITF�s

Multinucleated OsteoclastsMononuclear

Osteoclast

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Wnt signaling pathway schematic

Wnt proteins are highly-conserved secreted molecules that regulate cell-cell interactions during embryogenesis

Wnt signaling pathway is linked to several diseases including cancer as well as bone lesions and osteoporosis

Wnt signaling pathways consist of the canonical pathway, the Wnt-Ca2+ pathway, and the planar cell polarity pathway

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Key steps in pathway

Wnt inactive Wnt active

β-catenin is phosphorylatedby APC/axin/GSK-3βcomplex

Phosporylated β-catenin is degraded

Initiated when Wnt binds to Frizzled and LRP

Concentration of APC/axin/GSK-3β decreases and β-catenin accumulates

β-catenin/TCF complex initates transcription of Wnttarget genes

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Wnt signaling pathway regulation of bone

Wnt proteins involvement in bone (development and disease)

DKK-1 in MM with bone lesions, osteoporosis

LRP5 for osteoporosis (HBM phenotype)

Mechanism elucidation

Helps determine fate of mesenchymal stem cells

Increase of OPG/RANKL ratio leads to osteoclastogenesissuppression

Increases rate of proliferation and differentiation of osteoblasts

Inhibits rate of apoptosis of osteoblasts

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Overview of pathway model

Model componentsDKK1, Wnt, other ligands as neededLRP5/6, Frizzled, Kremen2DisheveledAxin, GSK3β, APCTCF/ LEFphosphorylated forms and intermediate complexes

Data used to construct model: only published data usedProtein expression data to establish initial concentrations; directly measured or derivedKinetic experiments to determine KD or kon and koff; derive using experimental data and mathematical optimizationUncertainty and variability in parameters owing to different cell and tissue types addressed through sensitivity analyses

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Model simulations: binary outcome captured

DKK-1 absent DKK-1 present

Wnt signaling pathway modeling

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Bone Platform: motivation and model paradigm

Systemicoutputs

Bonemetrics

Bonecells

Signalingfactors

Systemicinputs

RANKLRANK

(x,t) (x,t)

! Bone remodeling is a key pathophysiological process; multiple new targets! Model needs to accept systemic inputs ! Model needs to predict changes in systemic outputs such as key biomarkers! Model needs to link to clinical endpoints! Existing nonlinearities and different time scales

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RANK-RANKL-OPG system

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Model by Lemaire et al (J. Theor. Biol., 2004)

Responding OB

Active OC

OC Precursors

OPG

RANKApoptotic OB

synthesis

PTH

Active OB RANKL

PTH TGFβ

Progenitors Apoptotic OC

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Expanded bone model

Apoptotic OB

Responding OB

Active OC

T CellsBiomarker

TGFβmarkers

Active OB

Progenitors

Plasma Cells

Apoptotic OC

OC Precursors

OPG

DKK1

PTHβCatenin

Bone Cells Mass

Drug

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Bone model application

DKK-1 neutralizing antibodyMultiple myeloma with bone lesionsOsteoporosis

DKK-1 has been suggested as possible patient stratification biomarkerQuestions to be addressed by model:

Guide dosing based on levels of predicted free DKK-1Sensitivity of patients to treatment based on their existing osteoblast numbersCompare to existing therapies; assess combinations

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Clinical applications

Dose will be given when free DKK-1 level goes above a certain level

No assay measuring free DKK-1 is available

Assay measuring total DKK-1 is available

Calibrate the model based on free level of DKK-1 at baseline (for a range of subjects) and then use model to predict time courses of bound, free and total levels of DKK-1 for different dose levels of antibody

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Final PK/PD Model

PD predictions

PK predictions

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Model Predictions for Representative Patient

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Summary of results

Model can guide clinical therapy by predicting levels of free DKK-1Model suggests that hypothesized presence of a DKK-1 negative

feedback loop, i.e. Wnt-signaling and β-catenin translocation leads to DKK-1 up-regulation by osteoblasts (Gonzalez-Sancho et al., Nida et al) is true.

Model can quantify impact of disease severityModel output ultimately consists of BM

Effectiveness of therapyComparison with existing and planned therapies

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Thank you!

Questions?