Kwee Yong, UCL Cancer Institute multiple myeloma: D-type cyclin and PI3k pathways UKMF Spring meeting 2015 Kwee Yong, UCL Cancer Institute

Not one, but many myelomas

Dysregulation of D-type cyclin in multiple myeloma

Early oncogenic events dysregulate a cyclin D gene

Control of the Mammalian Cell cycle G0 p15 p16 p18 p19 M G1 Cyclin B-CDK1 pRb P Cyclin D1-CDK4/6 Cyclin D2 Cyclin D3 G2 E2F S What is CDC Cyclin D1: AP-1 site, STAT3 D2: myc The cyclin D-CDK4/6 complex phosphorylates retinoblastoma protein, relieving its repressor action, and allowing the transcription of genes required for DNA replication, mitosis etc pRb P E2F p21 p27 p57 Cyclin E-CDK2 p21 P27 p57 Cyclin A-CDK2

Expression of D-type Cyclins and their Dependent Kinases in primary myeloma cells Normal BM Multiple Myeloma BM Multiple Myeloma Extra-Medullary Cyclin D1 Cyclin D2 CDK 4 CDK 6 Phospho-Rb (K4/6) Total pRb p27 Transcriptional regulation of cyclin D’s PCNA Actin Normal PCs did not express cell cycle regulators MM Samples from patients with early stage, or stable disease did not express cell cycle regulators Increased expression of cell cycle regulators in patients with progressive disease (progressed on induction, florid relapse)

What is the significance of cyclin D expression in myeloma? Contribution of cell cycle dysregulation to disease pathogenesis Does cyclin D1-expressing myeloma behave differently from D2-expressing disease?

What is the significance of cyclin D expression in myeloma? Contribution of cell cycle dysregulation to disease pathogenesis Does cyclin D1-expressing myeloma behave differently from D2 disease?

Proliferative rate is prognostic Thomas et al, Eur J Hematol, 2010

Cell cycle analysis of myeloma cells Myeloma cell line NCI-H929 Primary CD138+ Cells G2M – 1.1% 17% Ki67 S – 1.4% G0/G1 – 79% DNA Content <G1 – 18.5% Quinn et al, Blood 2011

Proliferative fraction of freshly isolated bone marrow CD138+ myeloma cells Smouldering MM = 4 De Novo MM = 30 Plateau = 8 Relapse = 88 Extramedullary disease = 9 Total = 139 Quinn et al, Blood 2011

How is cell cycling regulated in myeloma cells? External mitogens or growth factors Alteration (mutations, copy number changes, epigenetic marks) of genes encoding cell cycle regulatory proteins Alteration of pathways regulating cell cycle proteins (post-translational)

How is cell cycling regulated in myeloma cells? External mitogens or growth factors Dysregulation (mutations, copy number changes, epigenetic marks) of genes encoding cell cycle regulatory proteins Dysregulation of pathways regulating cell cycle proteins (post-translational)

What is the significance of cyclin D expression in myeloma? Contribution of cell cycle dysregulation to disease pathogenesis Does cyclin D1-expressing myeloma behave differently from D2 disease?

Cyclin D2 myeloma cells respond to cytokines Quinn et al, Blood 2011 n = 12 n= 14

Is Cyclin D1 Functional in t(11;14) MM Cells? Immuno-precipitation experiments indicate that cyclin D1 is found in complexes with CDK4/6 Smith D, unpublished data

Expression of cell cycle proteins in vivo: comparing cyclin D1 with D2 tumours CDK4 CDK6 Phospho-pRb Cyclin D1 Cyclin D2

Is Cyclin D1 Functional in t(11;14) MM Cells? Nonsilencing Cyclin D1 MAPK siRNA: - (6g) Cyclin D1 Phospho-pRb (K4/6) Total pRB 100% 90% G2/M S G1 <2n 80% 70% 60% % Cells in each Cell Cycle Phase 50% 40% 30% 20% 10% 0% - : siRNA (6g) Cyclin D1 Nonsilencing MAPK Glassford et al, Brit J Haem 2009

Why are cyclin D1 tumours less proliferative? G1 Cyclin B-CDC2 pRb P Cyclin D1-CDK4/6 Cyclin D2 Cyclin D3 G2 E2F S Cyclin D1/CDK2 complexes are inactive pRb P E2F p21 p27 p57 Cyclin E-CDK2 p21 P27 p57 Cyclin A-CDK2

Non-cell cycle functions of cyclin D1? DNA repair Survival Jirawatnotai et al, Nature 2011 Beltran et al, PNAS 2011

How is cell cycling regulated in myeloma cells? External mitogens or growth factors Dysregulation (mutations, copy number changes, epigenetic marks) of genes encoding cell cycle regulatory proteins Dysregulation of pathways regulating cell cycle proteins (post-translational)

Chromosome 1q amplification in myeloma Frequency increases with disease progression Associated with shorter survival Shah et al, ASBMT 2015 Avet-Loiseau, JCO 2012

Prognostic impact of chr 1q gain may be related to cell cycle effects of CKS1b Gain of 1q21 associated with increased expression of CKS1b Part of E3 ubiquitin ligase targeting p27 Associates with CDK2 to enhance kinase activity Net result is increase in cell cycling Freshly isolated CD138+ cells from patients with chr1q gain have higher levels of basal proliferation as measured by Ki67/PI Quinn J, unpublished data

Myeloma cells with Cks1b amp have lower levels of p27 Add effect of CKS1b k/d on p27 levels Primary CD138+ cells Quinn J, unpublished data

Therapeutic implications

Cell cycle Inhibitors CDK inhibitors Small molecule inhibitor of CDK4 and CDK6 In vitro and in vivo pre-clinical data Phase 1 study with Bortezomib & Dexamethasone Right spelling? Smith D, unpublished data

Cell cycle Inhibitors Dinaciclib: CDK inhibitor (CDK1, 2, 5, 9, also cyclin D/CDK4) Phase 1 study in RRMM completed 11% PR, 19% MR or better PFS 3.5 mo, OS 18.8 mo Right spelling?

How is cell cycling regulated in myeloma cells? External mitogens or growth factors Alteration (mutations, copy number changes, epigenetic marks) of genes encoding cell cycle regulatory proteins Alteration of pathways regulating cell cycle proteins (post-translational)

What does PI3k signalling have to do with D-type cyclins and cell cycle control?

PI3k and mTOR signalling IL-6, IGF-1, BAFF, APRIL, Integrin binding GRB10 IRS1 PI3K PIP2 RAS PIP3 INFLAMMATION RAF/MEK PTEN ERK/RSK IKKB mTORC2 PDK1 AMP:ATP AMPK SGK TSC1/2 AKT S473 T308 Amino acids Rag A/B Rag C/D GSK3 RHEB PRAS40 mTORC1 FOXOs MDM2 PI3k pathway activated by cytokine binding to RTK, leading to auto-phosphorylation and phosphorylated tyrosine residue acts as docking site for p85, regulatory sub-unit of PI3k, that recruits p110, catalytic subunit to the membrane. Ras can also induce membrane localisation and activation of p110. Cyclin D1 is phosphorylated by GSK3, to induce rapid degradation and protein turnover. Inhibition of GSK3 by Akt phosphorylation P21 is phosphorylated by Akt and this enhances its binding to, and activation of cyclin D/CDK4/6 complexes, while reducing binding to and inhibition of cyclin E/CDk2. p21 also has a role in survival. Akt blocks FOXO-mediated activation of p27, P27 is also directly phosphorylated by Akt, leading to retention in the cytoplasm and MYC S6K1 4EBP1 p21 Cyclin D1 p27 BIM BAD p53 S6 eIF4E ULK1 ATG13 Proliferation and Survival Cell growth Autophagy

PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 GRB10 IRS1 PI3K PIP2 RAS PIP3 INFLAMMATION RAF/MEK PTEN AKTi Akti1/2, AZD5363, MK2206 ERK/RSK IKKB mTORC2 PDK1 AMP:ATP AMPK mTORi PP242, KU006, WYE-354 SGK TSC1/2 AKT S473 T308 Amino acids Rag A/B Rag C/D GSK3 RHEB PRAS40 MDM2 mTORC1 FOXOs Rapamycin PI3k pathway activated by cytokine binding to RTK, leading to auto-phosphorylation and phosphorylated tyrosine residue acts as docking site for p85, regulatory sub-unit of PI3k, that recruits p110, catalytic subunit to the membrane. Ras can also induce membrane localisation and activation of p110. Cyclin D1 is phosphorylated by GSK3, to induce rapid degradation and protein turnover. Inhibition of GSK3 by Akt phosphorylation P21 is phosphorylated by Akt and this enhances its binding to, and activation of cyclin D/CDK4/6 complexes, while reducing binding to and inhibition of cyclin E/CDk2. p21 also has a role in survival. Akt blocks FOXO-mediated activation of p27, P27 is also directly phosphorylated by Akt, leading to retention in the cytoplasm and MYC S6K1 4EBP1 p21 Cyclin D1 p27 BIM BAD p53 S6 eIF4E ULK1 ATG13 Proliferation and Survival Cell growth Autophagy

Activation of PI3K pathway in myeloma is not universal, may depend on D-type cyclin / IgH Tx pAkt S473 pS6 S235/236 p4EBP1 T37/46 GAPDH KMS27 KMS21 U266 JIM1 LP1 H929 OPM2 KMS11 R8226 JJN3 MM1S KMS12BM t(4;14) t(14;16) t(11;14) Phospho-Akt by flow *** *** t(14;16) t(4;14) t(11;14) Zollinger et al, Blood 2008 Stengel et al, Leukemia 2012

Effect of dual PI3K/mTOR blockade on growth of myeloma cells MM1.s Cyclin D2 NCI-H929 Cyclin D2 KMS12BM Cyclin D1 t(11;14) t(14;16) t(4;14) PI-103 (mmol)

Dual PI3K/mTOR blockade decreases cell cycling in cyclin D2 cells IGF-I IGF-I + PI103 MM1-S KMS-12BM <2n G1 S G2/M Control Cyclin D2 Cyclin D1

PI3K signalling regulates cell cyclin D2 but not cyclin D1 MM1-S PI103: - + - + - + - + control FCS IGF-I IL-6 KMS12-BM Cyclin D1 phospho-pRb CDK6 p27 CDK4 Total pRb Actin Cyclin D2 PI103: - + - + - + - + control FCS IGF-I IL-6

PI3k/mTOR blockade reduces proliferation in primary MM cells expressing cyclin D2 t(14;16) t(4;14) Patient # 5 Patient # 6 Patient # 7 Patient # 8 Patient # 9 Patient # 10 Patient # 11 Patient # 12 Cyclin D2 IGF-I: - + + - + + PI-103: - - + - - + phospho-Rb (K4/6) Patient # 9 Patient # 8 Actin

Effect of PI3K/mTOR blockade on survival of MM cells HMCL CD138+ BM cells 0h 4h 8h 24h PI ANN V 6% 11% 84% 7% 23% 70% 10% 67% 25% 44% 30%

PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 GRB10 IRS1 PI3K PIP2 RAS PIP3 INFLAMMATION RAF/MEK PTEN AKTi Akti1/2, AZD5363, MK2206 ERK/RSK IKKB mTORC2 PDK1 AMP:ATP AMPK mTORi PP242, KU006, WYE-354 SGK TSC1/2 AKT S473 T308 Amino acids Rag A/B Rag C/D GSK3 RHEB PRAS40 MDM2 mTORC1 FOXOs Rapamycin PI3k pathway activated by cytokine binding to RTK, leading to auto-phosphorylation and phosphorylated tyrosine residue acts as docking site for p85, regulatory sub-unit of PI3k, that recruits p110, catalytic subunit to the membrane. Ras can also induce membrane localisation and activation of p110. Cyclin D1 is phosphorylated by GSK3, to induce rapid degradation and protein turnover. Inhibition of GSK3 by Akt phosphorylation P21 is phosphorylated by Akt and this enhances its binding to, and activation of cyclin D/CDK4/6 complexes, while reducing binding to and inhibition of cyclin E/CDk2. p21 also has a role in survival. Akt blocks FOXO-mediated activation of p27, P27 is also directly phosphorylated by Akt, leading to retention in the cytoplasm and MYC S6K1 4EBP1 p21 Cyclin D1 p27 BIM BAD p53 S6 eIF4E ULK1 ATG13 Proliferation and Survival Cell growth Autophagy

PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 Buparlisib PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 GRB10 IRS1 PI3K PIP2 RAS PIP3 INFLAMMATION RAF/MEK PTEN AKTi Akti1/2, AZD5363, MK2206 ERK/RSK IKKB mTORC2 PDK1 AMP:ATP AMPK mTORi PP242, KU006, WYE-354 SGK TSC1/2 AKT S473 T308 Amino acids Rag A/B Rag C/D GSK3 RHEB PRAS40 FOXOs MDM2 mTORC1 Rapamycin PI3k pathway activated by cytokine binding to RTK, leading to auto-phosphorylation and phosphorylated tyrosine residue acts as docking site for p85, regulatory sub-unit of PI3k, that recruits p110, catalytic subunit to the membrane. Ras can also induce membrane localisation and activation of p110. Cyclin D1 is phosphorylated by GSK3, to induce rapid degradation and protein turnover. Inhibition of GSK3 by Akt phosphorylation P21 is phosphorylated by Akt and this enhances its binding to, and activation of cyclin D/CDK4/6 complexes, while reducing binding to and inhibition of cyclin E/CDk2. p21 also has a role in survival. Akt blocks FOXO-mediated activation of p27, P27 is also directly phosphorylated by Akt, leading to retention in the cytoplasm and MYC S6K1 4EBP1 p21 Cyclin D1 p27 BIM BAD p53 S6 eIF4E ULK1 ATG13 Proliferation and Survival Cell growth Autophagy

Phase 1B Study of Buparlisib with Bortezomib in Defined Genetic Subgroups of Patients with Relapsed or Refractory Multiple Myeloma Primary OBJECTIVES To determine the MTD (and/or RP2D, recommended phase 2 dose) of buparlisib with bortezomib (BKM-Bz) in relapsed/refractory multiple myeloma (MM) patients (dose escalation part) To evaluate the safety of the combination of BKM-BZ in patients with relapsed/refractory MM (dose expansion part)

Summary and Conclusions Cyclin D1 and D2 tumours differ In cell cycle response to mitogens Dependence on PI3k pathway for proliferation and survival Exploit such differences by tailoring therapies to particular genetic sub-types Understand how newly acquired genetic lesions impact on the cellular wiring imposed by type of D cyclin