Advances in Biology and Pathophysiology of Multiple Myeloma

1. Advances in Biology and Pathophysiology of Multiple Myeloma Amer G. Rassam, MD

2. History of Multiple Myeloma • First case, a London grocer “Thomas Alexander McBean” • Jumped from a cave in 1844 • According to Drs. Thomas Watson and William MacIntyre, Mr. McBean had “Mollities et Fragilitas Ossium” • Mr. McBean died on New Year’s day in 1846

3. History of Multiple Myeloma • Urine sample presented to “Henry Bence Jones” • Large amount of protein was found in the sample • The protein has became known as Bence Jones Protein

4. History of Multiple Myeloma In 1890s, Paul Unna and Ramon Cajal identified the plasma cell as a cell type and the cause of Multiple Myeloma Paul Gerson Unna 1850-1929 Santiago Ramon Y. Cajal 1852-1934

5. History of Multiple Myeloma • In 1873, Rustizky introduced the name Multiple Myeloma • In 1922, Bayne-Jones and Wilson identified 2 distinct groups of Bence Jones protein • In 1956, Korngold and Lipari identified the relationship between Bence Jones protein and serum proteins

6. Epidemiology of Multiple Myeloma • Prevalence (at any one time) : 40000 • Incidence: 14000 diagnosed each year • Median age: 65 • Median survival: 33 months • M:F 53:47 • 1.1% of all cancer diagnosis • 2% of all cancer deaths

7. Age Distribution in Multiple Myeloma 35 30 25 20 % 15 10 5 0 <40 40-49 50-59 60-69 70-79 >80 Age

8. Monoclonal Gammopathies – Mayo clinic Macro 3% (30) Extramedullary 1% (8) Other 3% (33) SMM 4% (39) LP 3% (37) AL 8% (90) MGUS 62% (659) MM 16% (172)

9. Immunophenotype of Multiple Myeloma Marker Features

10. Normal B-cell Development Lymph Node Short-lived plasma cell :: ::... Lymphoplasmacyte (memory B Cell) IgM IgM Follicle center Lymphoblast :: Somatic Hypermutation of Ig Sequences Stimulation with Antigen Plasmablast Naïve B Cell Isotype Switching Bone Marrow ::... G, A, D, E Long-lived plasma cell Pre-B cell

11. Mechanisms of Disease Progression in Monoclonal Gammopathies Kyle RA et al. N Engl J Med. 2004 Oct 28;351(18):1860-73

12. Chromosomal Abnormalities in MM Translocations (listed in order of frequency)

13. Chromosome 13 Deletions in MM 11 12 13 14 21 22 31 32 33 34 Shaughnessy J et al, Blood, 2000; 96:1505

14. Pathogenesis of Multiple Myeloma Two pathways involved in the early pathogenesis of MGUS and MM 50% Hyperdiploid 50% non-hyperdiploid Infrequent IgH Translocations IgH Translocations 11q13 (cyclin D1) 4p16 FGFR3+ MMSET 6p21 (cyclin D3) Multiple trisomies of chromosomes 3, 5, 7, 9, 11, 15, 19 and 21 16q23 (c-maf) 20q11 (mafB) Hideshima et al, Blood, August 2004, 607-618

15. Pathogenesis of Multiple Myeloma 100 90 80 70 60 Prevelance of IgH Translocations 50 40 30 20 10 0 MGUS MM PPCL HMCLs Hideshima et al, Blood, August 2004, 607-618

16. Prevalence of IgH Translocations 4p16 or 16q23 • Lower incidence with MGUS/SMM • de novo MM • Rapid progression of MGUS to MM • Extremely poor prognosis

17. Translocations in MM Secondary Primary c-myc 6p21 11q13 20q11 15% MM 40% adv MM 90% HMCLs 4p16 16q23 Hideshima et al, Blood, August 2004, 607-618

18. Translocation and Cyclin D (TC) Molecular Classification of MM Bergsagel and Kuehl, Immunol Rev, 2003, 194:96-104

19. Cyclin D Expression in Normal and Malignant Plasma Cells D1=Green,D2=Red,D3=Blue PPC BMPC 6p 11q13 D1 D1+D2 other 4p16 maf TC1 TC2 TC3 TC4 TC5 Tarte k. et al, Blood. 2002;100:1113-1122. Zhan F. et al, Blood. 2002; 99:1745-1757

20. Dysregulation of cyclin D1, D2, D3 “a unifying oncogenic event in MM” • MGUS and MM appear closer to normal PCs than to normal PBs • >30% of cells can be in S phase • Expression level of cyclin D1, D2, D3 mRNA in MM and MGUS is distinctly higher than normal PCs • Expression level of cyclin D2 mRNA is comparable with that expressed in normal proliferating PBs

21. Dysregulation of cyclin D1, D2, D3 “a unifying oncogenic event in MM” • Cyclin D1 is not expressed in normal hemopoitic cells • Cyclin D1 expressed in 40% of MM lacking a t(11;14) translocation • Ig translocations that dysregulate cyclin D1 or D3 occur in about 20% of MM tumors • Therefore, almost all MM tumors dysregulate at least one of the cyclin D genes

22. p18 N, K-RAS DEL 13 c-myc ?p16 FGFR3 p53 Progression to Plasma Cell Neoplasia Germinal center B cell Intramedullary Myeloma Extramedullary Myeloma MGUS HMCL 11q13 6p21 NON-HYPER DIPLOID 16q23 Primary IgH tx 20q11 4p16 Other TRISOMY 3, 5, 7, 9, 11, 15, 19, 21 HYPER DIPLOID Hideshima et al, Blood, August 2004, 607-618

23. Progression to Plasma Cell Neoplasia Normal Plasma Cell Intra- medulary myeloma Extra- medullary myeloma MGUS IgH translocations Deletion of 13q Chromosomal instability RAS mutations Dysregulation of c-MYC p53 mutations

24. The TC Molecular Classification Predicts Prognosis and Response to Therapies Increased PC Labeling Index Lack of Cyclin D1 Expression Deletion of p53 Monosomy of chro 13/13q Hypodiploidy Bad prognosis Activating Mutations of K-Ras Monosomy of chro 17 Tumor Cells with Abnormal Karyotype t(14;16) TC5 t(4;14) TC4

25. The TC Molecular Classification Predicts Prognosis and Response to Therapies t(4;14) translocation (TC 4) Shortened Survival Standard Therapy (42) High-dose Therapy (22) Median OS 26 months Median OS 33 months Fonseca R et al, Blood. 2003; 101:4569-4575 Moreau et al, Blood. 2002; 100:1579-1583

26. The TC Molecular Classification Predicts Prognosis and Response to Therapies t(14;16) translocation (TC 5) Shortened Survival (worse Prognosis) Standard Therapy (15) Median OS 16 months Fonseca R et al, Blood. 2003; 101:4569-4575

27. The TC Molecular Classification Predicts Prognosis and Response to Therapies t(11;14) translocation (TC 1) Better Survival Standard Therapy (53) High-dose Therapy (26) Median OS 50 months Median OS 80 months Fonseca R et al, Blood. 2003; 101:4569-4575 Moreau et al, Blood. 2002; 100:1579-1583

28. The TC Molecular Classification Predicts Prognosis and Response to Therapies • The TC classification may be clinically useful way to classify patients into groups that have distinct subtypes of MM (and MGUS) tumors. • The TC classification identifies clinically important molecular subtypes of MM with different prognosis and with unique responses to different treatments.

29. The TC Molecular Classification Predicts Prognosis and Response to Therapies • High dose therapy and TC1 • Microenvironment-directed therapy and TC2 • FGFR3 inhibitor and TC4 • maf dominant-negative and TC5

30. p p p p Critical role for Cyclin D/Rb pathway in MM TC1 TC5 TC4 TC3 TC2 Hyperdiploid 11q13 CCND1 16q23 c-maf 4p16 Other Cyclin D1 6p21 CCND3 20q11 mafB FGFR3 MMSET p16 INK4a p15 INK4b Cyclin D2 Cyclin D1 Cyclin D3 p18 INK4c CDK 4, 6 CDK 4, 6 CDK 4, 6 p19 INK4d G1 Phase S Phase Rb E2F E2F Rb OFF ON

31. Novel Therapeutic Strategies targeting Genetic Abnormalities Targeting the genes Directly dysregulated By translocation Targeting Cyclin D Silencing of CDK inhibitor mRMA expression might be reversed Targeting FGFR3 by monoclonal antibodies Desferroxamine HDAC Inhibitors Targeting FGFR3 by selective tyrosine kinase inhibitor Selective CDK inhibitors DNA methyl Transferase inhibitor

32. Interaction of MM cells and their BM milieu GSK-3β FKHR Caspase-9 NF-KB mTOR Bad migration Survival Anti-apoptosis Cell cycle PKC Akt TNFα TGFβ VEGF IL-6 PI3-K Bcl-xL MCL-1 Survival Anti-apoptosis JAK/STAT3 MEK/ERK Proliferation Bcl-xL IAP Cyclin-D Survival Anti-apoptosis Cell cycle NF-KB IL-6 VEGF IGF-1 SDF-1α MEK/ERK Proliferation Anti-apoptosis p27Kip1 MM ERK Smad2 NF-KB Adhesion molecules LFA-1 ICAM-1 NF-KB BMSC MUC-1 VCAM-1 Fibronectin VLA-4

33. Myeloma Cells and BM Microenvironment Bruno et al, The Lancet Oncology, July 2004, 430-442

34. Apoptotic Signaling Pathways TNFα FasL TRAIL Velcade ZME-2 ImiDs, Velcade HDAC-I, 2ME-2 Dex JNK Mitochondria FADD Bid Cytochrome-c Smac IL-6 IGF-1 Caspase-8 Caspase-9 Caspase-3 PARP Apoptosis Hideshima et al, Blood, August 2004, 607-618

35. Novel biologically based therapies targeting MM cells and the BM microenvironment Novel Agents Apoptosis Growth Arrest A Adhesion Molecule B Inhibition of Adhesion Proliferation C bFGF VEGF Inhibition of Cytokines IL-6 IGF-1 VEGF SDF-1α D Angiogenesis Drug Resistance

36. Novel Agents for Myeloma • Targeting both MM cells and interaction of MM cells with the BM microenvironment • Targeting circuits mediating MM cell growth and survival • Targeting the BM microenvironment • Targeting cell surface receptors

37. Novel Agents for Myeloma Targeting both MM cells and their interaction with BM microenvironment Targeting circuits mediating MM cell growth and survival • Thalidomide and its analogs (Revlimid) • Proteasome inhibitor (Bortezomib) • Arsenic trioxide • 2-Methoxyestradiol (2-ME2) • Lysophosphatidic acid acyltransferase-β inhibitor • Triterpinoid 2-cyano-3, 12-dioxoolean-1, 9-dien-28- oic acid (CDDO) • N-N-Diethl-8, 8-dipropyl-2-azaspiro [4.5] decane-2-propanamine (Atiprimod) • VEGF receptor tyrosine kinase inhibitor (PTK787/ZK222584, GW654652) • Farnesyltransferase inhibitor • Histone deacetylase inhibitor (SAHA, LAQ824) • Heat shock protein-90 inhibitor (Geldanamycin,17-AAG) • Telomerase inhibitor (Telomestatin) • bcl-2 antisense oligonucleotide (Genasense) • Inosine monophophate dehydrogenase (VX-944) • Rapamycin Targeting the bone marrow microenvironment Targeting cell surface receptors • IĸB kinase (IKK) inhibitor (PS-1145) • p38 MAPK inhibitor (VX-745, SCIO-469) • TFG-β inhibitor (SD-208) • TNF related apoptosis-inducing ligand (TRAIL) / Apo2 ligand • IGF-1 receptor inhibitor ( ADW) • HMG-CoA reductase inhibitor (statins) • Anti-CD20 antibody (Rituximab)

38. Proposed Mechanism of Action of Drugs in Targeting Myeloma Cells and BM Microenvironment Kyle RA et al. N Engl J Med. 2004 Oct 28;351(18):1860-73

39. Homoeostasis of Healthy Bone Tissue and MM Bone Disease Bruno et al, The Lancet Oncology, July 2004, 430-442

40. Osteoprotegerin (OPG) Osteoblast T cell Osteoclast Precursor Bone Destruction Osteoclast Bone Marrow stromal Cells Interferon ɣ MIP1 TNFα IL1β IL7 IL6 RANKL Multiple Myeloma Cells RANK

41. Effects of Thalidomide on the Myeloma Microenvironment Bruno et al, The Lancet Oncology, July 2004, 430-442

42. Proposed Action of Thalidomide in Myeloma Mutiple Myeloma Cells Modulation of Cytokines VEGF IL6 TNFα IL1β Direct Action Bone Marrow Stromal Cells T Lymphocytes IL2 ILNɣ Modulation of Immune System Bone Marrow Vessels VEGF bFGF Inhibition of Angiogenesis Cytotoxicity of NK Cells

43. Mechanism of Action of Bortezomib • Phosphorylation of NFKB inhibitory partner protein IKB leads to degradation of IKB by the proteosome and release of NFKB • NFKB migrates into the nucleus to induce arrest of apoptosis and expression of adhesion molecule • Affinity of Bortezomib for the proteosome inhibits protein degradation, and prevents nuclear translocation of NFKB Bruno et al, The Lancet Oncology, July 2004, 430-442

44. Mechanism of Action of Arsenic Trioxide • Mutated P53: Arsenic trioxide triggers the caspase cascade by activation of caspases 8 and 10 • Functional P53: The cascade is activated through the mitochondrial apoptotic pathway and the activation of caspase 9 Bruno et al, The Lancet Oncology, July 2004, 430-442