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

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

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

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

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

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

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

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)

Immunophenotype of Multiple Myeloma Marker Features CD10 Subset CD19 & CD20 Rarely expressed CD28 & CD86 Occurs with progressive disease CD34 Not expressed by malignant clone CD38 High expression of most but not all malignant cells CD56 (N-CAM) Absent in MGUS and PCL CD138 Syndecan-1 is over expressed

Normal B-cell Development Long-lived plasma cell 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

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

Chromosomal Abnormalities in MM Translocations (listed in order of frequency) 14q32 with 11q13 (cyclin D, other new fibroblastic growth factors) 4p16 (FGFR3) 6p25 (Interferon regulatory factor 4) 16q23 (C-MAF transcription factor) 8q24 (C-MYC) 18q21 (BCL-2) 1q with 5, 8, 12, 14, 15, 16, 17, 19q, 21, 22 Losses 6q, 13q Gains 3, 5, 7, 9q, 11q, 12q, 15q, 17q, 18, 19, 21, 22q

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

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

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

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

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

Translocation and Cyclin D (TC) Molecular Classification of MM Group Primary translocation Gene(s) at breakpoint D-Cyclin Ploidy Freq of TC in newly diag MM, % TC1 11q13 6p21 CCND1 CCND3 D1 D3 NH NH 15 3 TC2 None D1 H 37 TC3 D2 H=NH 22 TC4 4p16 FGFR3/MMSET NH>H 16 TC5 16q23 20q11 c-maf mafB D2 D2 5 2 Bergsagel and Kuehl, Immunol Rev, 2003, 194:96-104

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

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

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

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 DEL 13 ?p16 p18 p53 c-myc N, K-RAS FGFR3 TRISOMY 3, 5, 7, 9, 11, 15, 19, 21 HYPER DIPLOID Hideshima et al, Blood, August 2004, 607-618

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

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

t(4;14) translocation (TC 4) 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

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

t(11;14) translocation (TC 1) 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

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.

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

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 p p p p Rb E2F E2F Rb OFF ON

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

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

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

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

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

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

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)

Kyle RA et al. N Engl J Med. 2004 Oct 28;351(18):1860-73 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

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

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

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

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

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

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