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MULTIPLE MYELOMA: D-type cyclin and PI3k pathways UKMF Spring meeting 2015 Kwee Yong, UCL Cancer Institute.

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Presentation on theme: "MULTIPLE MYELOMA: D-type cyclin and PI3k pathways UKMF Spring meeting 2015 Kwee Yong, UCL Cancer Institute."— Presentation transcript:

1 MULTIPLE MYELOMA: D-type cyclin and PI3k pathways UKMF Spring meeting 2015 Kwee Yong, UCL Cancer Institute

2 Not one, but many myelomas

3 Dysregulation of D-type cyclin in multiple myeloma

4 Early oncogenic events dysregulate a cyclin D gene

5 Control of the Mammalian Cell cycle Cyclin D1-CDK4/6 Cyclin D2 Cyclin D3 Cyclin E-CDK2 p15 p16 p18 p19 Cyclin A-CDK2 p21 P27 p57 G2 M S E2F pRb P P P G1 pRb P E2F p21 p27 p57 Cyclin B-CDK1 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 G0

6 Phospho-Rb (K4/6) Cyclin D2 Cyclin D1 CDK 4 CDK 6 Total pRb Normal BM Multiple Myeloma Extra-Medullary p27 PCNA Multiple Myeloma BM Expression of D-type Cyclins and their Dependent Kinases in primary myeloma cells 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) Actin

7 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?

8 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?

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

10 Ki67 DNA Content 17% Myeloma cell line NCI-H929 Primary CD138+ Cells G2M – 1.1% S – 1.4% G0/G1 – 79%

11 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

12 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)

13 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)

14 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?

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

16 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

17 Expression of cell cycle proteins in vivo: comparing cyclin D1 with D2 tumours CDK4CDK6Phospho-pRb Cyclin D1 Cyclin D2

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

19 Cyclin D1-CDK4/6 Cyclin D2 Cyclin D3 Cyclin E-CDK2 p15 p16 p18 p19 Cyclin A-CDK2 p21 P27 p57 G2 M S E2F pRb P P P G1 pRb P E2F p21 p27 p57 Cyclin B-CDC2 Why are cyclin D1 tumours less proliferative? Cyclin D1/CDK2 complexes are inactive

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

21 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)

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

23 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

24 Myeloma cells with Cks1b amp have lower levels of p27 Primary CD138+ cells Quinn J, unpublished data

25 Therapeutic implications

26 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 Smith D, unpublished data

27 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

28 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)

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

30 PIP2 PIP3 PI3K PTEN AKT PDK1 mTORC2 T308 S473 mTORC1 4EBP1 eIF4E S6K1 S6 Cell growth IRS1 GRB10 TSC1/2 RHEB ERK/RSK IKKB AMPK Rag A/B Rag C/D Amino acids AMP:ATP ULK1 ATG13 Autophagy PRAS40 FOXOs SGK p27 BIM MDM2 p53 GSK3 Cyclin D1 MYC Proliferation and Survival BAD INFLAMMATION RAS RAF/MEK p21 IL-6, IGF-1, BAFF, APRIL, Integrin binding PI3k and mTOR signalling

31 PIP2 PIP3 PI3K PTEN AKT PDK1 mTORC2 T308 S473 mTORC1 4EBP1 eIF4E S6K1 S6 Cell growth IRS1 GRB10 TSC1/2 RHEB ERK/RSK IKKB AMPK Rag A/B Rag C/D Amino acids AMP:ATP ULK1 ATG13 Autophagy PRAS40 FOXOs SGK p27 BIM MDM2 p53 GSK3 Cyclin D1 MYC Proliferation and Survival BAD INFLAMMATION RAS RAF/MEK p21 PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 AKTi Akti1/2, AZD5363, MK2206 mTORi PP242, KU006, WYE-354 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 Rapamycin

32 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 KMS27KMS21 U266 JIM1 LP1 H929 OPM2 KMS11 R8226 JJN3 MM1S KMS12BM t(4;14)t(14;16)t(11;14) Phospho-Akt by flow Zollinger et al, Blood 2008 Stengel et al, Leukemia 2012 t(4;14) t(14;16) t(11;14) ***

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

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

35 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 phospho-pRb CDK6 PI103: control FCS IGF-I IL-6 CDK4 p27 Total pRb Actin

36 PI3k/mTOR blockade reduces proliferation in primary MM cells expressing cyclin D2 Cyclin D1 t(11;14) t(14;16) t(4;14) Patient # 5 Patient # 6 Patient # 7 Patient # 8 Patient # 9 Patient # 10 Patient # 11 Patient # 12 P<0.01 P<0.02 Cyclin D2 IGF-I: PI-103: phospho-Rb (K4/6) Patient # 9Patient # 8 Actin Cyclin D2

37 Effect of PI3K/mTOR blockade on survival of MM cells 0h 4h 8h 24h PI ANN V 6% 11% 84% 7% 23% 70% 10% 23% 67% 25% 44% 30% HMCLCD138+ BM cells

38 PIP2 PIP3 PI3K PTEN AKT PDK1 mTORC2 T308 S473 mTORC1 4EBP1 eIF4E S6K1 S6 Cell growth IRS1 GRB10 TSC1/2 RHEB ERK/RSK IKKB AMPK Rag A/B Rag C/D Amino acids AMP:ATP ULK1 ATG13 Autophagy PRAS40 FOXOs SGK p27 BIM MDM2 p53 GSK3 Cyclin D1 MYC Proliferation and Survival BAD INFLAMMATION RAS RAF/MEK p21 PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 AKTi Akti1/2, AZD5363, MK2206 mTORi PP242, KU006, WYE-354 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 Rapamycin

39 PIP2 PIP3 PI3K PTEN AKT PDK1 mTORC2 T308 S473 mTORC1 4EBP1 eIF4E S6K1 S6 Cell growth IRS1 GRB10 TSC1/2 RHEB ERK/RSK IKKB AMPK Rag A/B Rag C/D Amino acids AMP:ATP ULK1 ATG13 Autophagy PRAS40 FOXOs SGK p27 BIM MDM2 p53 GSK3 Cyclin D1 MYC Proliferation and Survival BAD INFLAMMATION RAS RAF/MEK p21 PI3k and mTOR signalling PI3Ki PIK90, GDC0941, ZSTK474 AKTi Akti1/2, AZD5363, MK2206 mTORi PP242, KU006, WYE-354 PI3K+mTORi (dual inhibitors) PI103, BEZ235, XL765 Rapamycin Buparlisib

40 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) Phase 1B Study of Buparlisib with Bortezomib in Defined Genetic Subgroups of Patients with Relapsed or Refractory Multiple Myeloma

41 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


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