Survival signalling in myeloma ucl cancer institute Survival signalling in myeloma Asim Khwaja UCL Cancer Institute & Department of Haematology

Rationale for developing targeted therapies - we are likely to be close to the limits of what can be achieved with conventional cytotoxic drugs

Today’s talk Background Potential targets for therapeutic intervention PI3-Kinase and mTOR PIM kinases

The molecular pathology of myeloma Kuehl & Bergsagel, JCI 2012

Myeloma microenvironment Kuehl & Bergsagel, JCI 2012

Potential signalling pathway targets in myeloma Microenvironment Molecular NF-kappa B TNF, BAFF, APRIL Dels/amp/mut of TRAF3, CYLD, BIRC2/3, NIK, NFKB1/2 etc RAS IGF1, IL6, FGF, chemokines Activating mutations NRAS, KRAS, BRAF, FGFR3 PI3-kinase IGF1, IL6, chemokines, integrins etc PTEN del, PIK3CA mut, RAS mutation, (DEPTOR overexpression) JAK kinase IL-6 WNT Multiple WNTs NOTCH Jagged, Delta-like

NF-kappaB signalling Canonical Alternative TNF BAFF, APRIL Proteasome TRADD TRAF2 TRAF3 TRAF2 TAK1 RIP1 cIAP1/2 NIK IKKg IKKa IKKa IKKb Proteasome IkB p100 RelB p50 p65 degradation processing p52 RelB p50 p65 p52 RelB

Primary MM cells display constitutive NF-κB activity that is largely resistant to high concentrations of bortezomib. Primary MM cells display constitutive NF-κB activity that is largely resistant to high concentrations of bortezomib. A. Representative mini-EMSA, using whole-cell lysates from the indicated total cell numbers, fold induction as determined by phosphoimager is labeled below the gel. B. Analysis of percent proteasome inhibition by increasing doses of bortezomib in CD138+ cells obtained from two MM patients using Proteo-Glo assay. Basal proteasomal activity for each cell type is assigned as 100% activity. C. EMSA analysis of lysates derived from CD138+ cells isolated from the indicated patients and then treated with 100 nmol/L bortezomib for 4 h where indicated. Fold change in NF-κB activity corrected for Oct-1 DNA binding is displayed below the gels. D. Fold change NF-κB DNA binding, as measured by phosphoimage quantification corrected for Oct-1 DNA binding, induced by treatment with 4 h of 100 nmol/L bortezomib in CD138+ cells from the indicated MM patients. Markovina S et al. Mol Cancer Res 2008;6:1356-1364 ©2008 by American Association for Cancer Research

Possible mechanism whereby bortezomib triggers canonical NF-κB activation. Possible mechanism whereby bortezomib triggers canonical NF-κB activation. Bortezomib either directly or indirectly (via RIP2) activates IKKβ, which subsequently phosphorylates IκBα, an inhibitor of p50/p65. After nonproteasomal degradation of IκBα, p50/p65 translocates to nucleus. IKKβ inhibitors block down-regulation of IκBα and NF-κB activity, as well as enhance bortezomib-induced cytotoxicity. Hideshima T et al. Blood 2009;114:1046-1052 ©2009 by American Society of Hematology

AKT PI3K, AKT and mTOR signalling PI3K PIP2 PIP3 PTEN Other targets GRB10 IRS1 PI3K PIP2 RAS PIP3 INFLAMMATION RAF/MEK PTEN ERK/RSK mTORC2 Other targets eg RAC IKKB PDK1 AMP:ATP AMPK SGK TSC1/2 AKT S473 T308 Amino acids Rag A/B Rag C/D GSK3 RHEB PRAS40 mTORC1 FOXOs MDM2 S6K1 4EBP1 p27 BIM BAD p53 Cyclin D1 MYC S6 eIF4E ULK1 ATG13 Proliferation and Survival Cell growth Autophagy

Akt Signaling GPCR C 2007-2009 SABiosciences.com Growth Factors Integrins Cytokines RT K Cytokine Receptor Ag GPCR BCR BCR G A B A (A)R PIP2 PIP3 IRS1 Ras GAB1 PI3K PI3K FAK Paxillin JAK1 PTEN GAB2 ILK BCAP Ga GTP Gb Gg PI3K PI3Kg CTMP PI3K Synaptic Signaling SYK PIP3 PIP3 PDK-1 PDK-1 Akt GLUT4 Vesicle Translation Regulation of Cyclic Nucleotide Caspase Cascade Elevation of Glucose Import p70S6K mTOR TSC2 PDE3B P HSP90 TSC1 Caspase9 P PFK1 PFK2 P CDC37 PP2A DNA-PK Glucose Uptake ERK Pathway Raf1 IKKs P 4EBP1 NF-kB Pathway Blocks Apoptosis Ser87 XIAP P Akt P WNK1 P BAD P Insulin Stimulated Mitogenesis eIF4E 14-3-3 BAD P Protein Synthesis Chk1 PRAS40 14-3-3 Apoptosis Inhibition Genetic Stability p21(CIP1) P p47Phox Cell Survival Respiratory Brust Cell Cycle Progression P p27(KIP1) YAP 14-3-3 14-3-3 14-3-3 P Htt p73 Mediated Apoptosis FKHR P CREB P Ataxin 14-3-3 MDM2 P AR P Neuroprotection ASK1 P GSK3 JIP1 Aggregation and Neurodegeneration Death Genes Cell Survival FKHR Glycogen Synthase CcnD eNOS P JNK Pathway CREB P Cell Cycle NO Production MDM2 P Glycogen Synthesis Ub Survival Genes p53 Ub p53 Degradation Cardiovascular Homeostasis C 2007-2009 SABiosciences.com Nucleus

Targeting PI3K, Akt and mTOR PI3Ki PIK90, GDC0941, ZSTK474 AKTi Akti1/2, AZD5363, MK2206 mTORi PP242, KU006, WYE-354 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765

Increased PI3K activity is associated with distinct myeloma IgH translocation categories Phospho-Flow Akt t(4;14) t(14;16) t(11;14) *** *** t(14;16) t(4;14) t(11;14) pAKT PIP3 LEVELS t(4;14) t(14;16) t(11;14) undetectable

Increased PI3K activity correlates with high level of cell death induced by dual PI3K/mTOR inhibition *** *** t(14;16) t(4;14) t(11;14) PI103 mTOR PI3K AKT Patient samples r=0.813, p=0.0001

Optimal cell killing requires inhibition of both PI3K and mTOR PI103 Bez235 PIK90 Rapa/Evero mTOR PP242 etc PI3K AKT Akti1/2

Potent feedback activation of PI3K by inhibitors of mTOR in myeloma cells * RAPA * *** pAktS473 pAktT308 mTORC1 AKT pS6 PIP3 (pmol/million cells) tubulin 8 24 8 24 8 24 8 24 8 24 CON PI PP1 PP2 PP5 PIP3 LEVELS PP242 mTORC2

There is significant PI3K-independent mTOR activity in myeloma cells ERK/RSK IKKB CON PI103 PIK90 BEZ RAP P90+RAP AMPK pAkt S473 mTORC1 AKT pS6 p4EBP1 tubulin 4 8 4 8 4 8 4 8 4 8 4 8 hours Cell growth Proliferation and Survival PI103 Bez235 PIK90 Rapa mTOR PI3K AKT

The role of DEPTOR in modulating PI3K and mTOR signalling in myeloma ERK/RSK PI3K IKKB Deptor GAPDH AMPK mTORC1 AKT DEPTOR Cell growth Proliferation and Survival HMCL DEPTOR pS6 p4EBP1 tubulin

PI3K/mTOR in myeloma There is a significant PI3K-independent component leading to mTOR activation Dual inhibition of PI3K and mTOR kinase activities induces maximal cytotoxic effects PI3K activity correlates with response to dual inhibitors Cytogenetics may predict pathway activation and hence response Evidence for synergy with glucocorticoids The role of DEPTOR is unclear

Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma Single and combined blockade of MEK1/2 and Akt in MM cell lines (n = 11). (A) MM cell lines were treated with the MEK1/2 inhibitor PD184352 (5 μmol/l), Akt inhibitor Akti‐1,2 (5 μmol/l), or their combination and cell survival was measured by flow cytometry. Whereas inhibition of MEK had only minor effects on cell survival, inhibition of Akt induced apoptosis, but with broad variance. Combination treatment significantly enhanced overall cell death, but still distinguished between resistance and sensitivity, marking the RAS‐mutated fraction as generally susceptible. (B) Titration experiments to show abrogation of phospho‐Erk1/2 and phospho‐Akt (Ser473) signals by suitable pharmacological inhibitors as assessed by Western blotting. (C) Cell lines harbouring oncogenic RAS (KRAS or NRAS point mutations at codons 12, 13, or 61) were always sensitive to combination treatment. Resistant/resilient cell lines (AMO‐1, U266, KMS‐11) were RAS wildtype (wt). DMSO, dimethyl sulfoxide; rel., relative; mut, mutated. © This slide is made available for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party. Steinbrunn et al British Journal of Haematology Volume 159, Issue 4, pages 430-440, 17 SEP 2012 DOI: 10.1111/bjh.12039 http://onlinelibrary.wiley.com/doi/10.1111/bjh.12039/full#bjh12039-fig-0002

Combined targeting of MEK/MAPK and PI3K/Akt signalling in multiple myeloma Single and combined pharmacological blockade of MEK1/2 and PI3K/mTOR in primary MM samples (n = 45) co‐cultured with bone marrow stromal cells. (A) As described in Fig , single‐agent MEK inhibition with PD0325901 had only minor effects on cell survival. Single‐agent PI3K/mTOR inhibition with 2 μmol/l PI103 overall induced a medium‐sized decrease in cell survival. Combined MEK1/2 and PI3K/mTOR inhibition significantly enhanced overall cell death induction. Whereas the group of samples sensitive for combination treatment contained both RAS wildtype (wt) and RAS‐mutated (mut) samples, the resistant subgroup was universally RAS wildtype. (B) Peripheral blood mononuclear cells were largely resistant to single‐agent as well as combined treatment with MEK1/2 and PI3K/mTOR inhibitors. (C) Exemplary Western analyses to show abrogation of Akt phosphorylation in primary MM cells after PI103 treatment either with and without presence of bone marrow stromal cells. D: DMSO, dimethyl sulfoxide; PI: PI103; rel., relative. © This slide is made available for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party. Steinbrunn et al British Journal of Haematology

Some conclusions Targeting single cell signalling pathways unlikely to be effective Activity of various combinations PI3K+MEK PI3K+mTOR PI3K/AKT+PIM Identification of biomarkers mutation analysis cytogenetics as surrogate expression of signalling proteins Integration with existing therapies

Acknowledgements Chloe Stengel Koremu Meja Kwee Yong Ching Cheung ucl cancer institute Acknowledgements Chloe Stengel Koremu Meja Ching Cheung Clare Shepherd Lolly Banerjee Kwee Yong