CLINICAL IMPLICATIONS OF TNF FAMILY IN BLOOD DISORDERS

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CLINICAL IMPLICATIONS OF TNF FAMILY IN BLOOD
DISORDERS

• DHEERAJ MOHANIA

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HISTORY

• Tumor necrosis factor (TNF), originally
  identified as a factor produced in the serum of
  endotoxin-injected animals, is a cytokine that
  mediates tumor necrosis.
• To date, 20 different members of the TNF
  superfamily and 21 different receptors have been
  identified.
• All ligands of the TNF superfamily have been
  found to activate transcription factor NF-?B and
  c-Jun kinase.
• Members of this family have diverse biologic
  effects, including induction of apoptosis,
  promotion of cell survival, and regulation of the
  immune system.
• The CD30,CD40,RANK and TRAIL are the four members
  that play important roles in regulating
  hematopoietic cells and are involved in the
  pathogenesis of several hematologic malignancies.

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TNF

• TNF ß
• Also called lymphotoxin
• produced by TH1 and Tc cells
• Target tissue tumour cells and macrophage and
  neutrophils
• Has cytotoxic activity and other effects similar
  to TNF a
• Enhances the phagocytic activity.

• TNF a
• Also called cachectin
• Produced by macrophages and
• Mast cells
• TNF alpha is a pleiotropic inflammatory cytokine
• Target tissue tumour cells and Inflammatory
  cells
• Has cytotoxic effects
• Induce cytokine secretion
• Responsible for extensive weight loss (cachexia)
  associated with chronic inflammation.

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Structural changes of cells undergoing necrosis
or apoptosis
.
Active substances released
Self-digestion
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DEATH signals
CD95 ligand / Fas ligand
TNF-alpha
Death receptors, e.g. CD95
Death domains
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Binding to the receptor induces receptors to
cluster and trimerize
Death domains
After binding to ligand, receptors form trimers
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FADD (Fas associated death domain protein) is
recruited via its death domain
DED (death effector domain) of FADD recruits
pro-caspase 8 via its DED
DISC complex
FADD-CASP8 complex brings multiple pro-caspase 8
molecules in close proximity (induced
proximity aggregation results in their
cross-activation).  This is the DISC -complex.
Activated caspase-8 (a heterotetramer) is
released from DISC into the cytoplasm
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caspase-8 cleaves pro-apoptotic BID
protein. BID interacts with the pro-apoptotic
BCL2 relatives BAX and BAK
This amplifies apoptosis induction through the
cell-extrinsic pathway
Nature Reviews Cancer 2 420-430 (2002)
TARGETING DEATH AND DECOY RECEPTORS OF THE
TUMOUR-NECROSIS FACTOR SUPERFAMILY
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TNF-alpha can signal to NF-kB transcription
factors.
NF-kB factors are anti-apoptotic
(mostly)
inflammatory cytokines, chemokines,
immunoreceptors, and cell adhesion molecules
Activate pro-survival Bcl-Xl, IAPs
Activate pro-apoptotic FAS, FAS Ligand P53
One of the central mediators of the immune
response'.
Tissue specific decision

• 1. The combinatorial response of
  promoter/enhancer regions, and
• The selective activation and binding of
  individual NF-kB proteins

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TNF-alpha can signal to NF-kappaB transcription
factors.
Binding of TNF-alpha to TNFR
TRADD recruited
IKK kinase recruited
IKK phosphorylate IkB
P50/p65 (NF-kappaB) Free to transactivate
IkB degraded
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NF-kappaB pathway
TNF-alpha can signal to NF-kappaB transcription
factors.
In the cytoplasm, NF-kB is inhibited by IkB
(inhibiitor of NF-kappa B)
Upstream activating signal (e.g., binding of TNF
to its receptor) may cause phosphorylation of
IkB by IKK (IkB kinase).
IKB -- is one of the NF-kB target genes
provide the regulatory loop
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NF-kappa B is an anti-apoptotic factor
Via NF-kappaB TNF blocks its own cell death
potential
chemotherapy activates NF-kB within tumor cells
NF-kB inhibitors augment chemotherapy
many common synthetic (e.g., aspirin), and
traditional (e.g., green tea, curcumin) remedies
target, at least in part, the NF-kB signaling
pathway
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Tumor Necrosis Factor superfamily.

• Type II transmembrane proteins (N-terminal inside
  the cell, C-terminal outside the cell) that share
  sequence homology in the extracellular domain.
• The biologically active forms of these protein
  ligands are self-assembled protein trimers.
• These trimers do not share any sequence homology
  at the receptor binding site, but they do share
  2530 sequence homology at trimerization sites.
• Many of the TNFSF members are cleaved in a
  biologically active soluble form. Although the
  majority of these ligands bind to one specific
  receptor, few bind to more than one receptor.

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TNF RECEPTOR FAMILY
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Tumor Necrosis Factor Receptor superfamily

• TNFRSF is a group of type I proteins that share
  sequence homology in their extracellular domains,
  
• Characterized by 40amino acid cysteine-rich
  repeats.
• The intracellular domains of these receptors are
  short and do not share any sequence homology,
  indicating their diverse functions.
• The cytoplasmic tails of these receptors signal
  by interacting with two major groups of
  intracellular proteins TNF receptorassociated
  factors (TRAFs) and Death domain (DD)-containing
  proteins.
• There are at least six human TRAFs that can
  interact with TRAF-binding sites on the
  cytoplasmic tail of some of these receptors to
  initiate several signaling pathways, including
  mitogen- activated protein (MAP) kinases, AKT,
  and NF-B.
• TNF-family receptors that have a DD sequence in
  their cytoplasmic tail are called death
  receptors. The DDs in these receptors interact
  with the DDs in adaptor proteins, such as
  Fas-associated DD (FADD) and TNF
  receptorassociated DD (TRADD).The adaptor
  proteins also contain a death effector domain
  (DED) that can interact with the DED in
  procaspase-8, leading to a cascade of apoptotic
  signals.

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Interacting Proteins of the TNF/TNFR Superfamily
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Neoplastic
B
B
apoptosis
B
B
B
Genetic Hit
B
HSC
apoptosis
B
B
Normal
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Apoptosis failure leads to disease
Especially true for autoimmune diseases
that result from the persistence of autoreactive
T/B-cells.
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LYMPHOCYTIC T OR B CELL LINEAGE
MYELOID Granulocyte Monocyte Erythrocyte platelets

CLL ALL
AML CML
Hodgkins and non-Hodgkins
Burkitts and Burkitts-like lymphoma/small
noncleaved B cell lymphoma
Lymphoblastic lymphoma
Diffuse large cell lymphoma (B/T/nul)
Anaplastic large cell lymphoma
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Cellular Origins of Lymphomas / Leukemias
PLEURIPOTENT STEM CELL
ACUTE LEUKEMIAS
LYMPHOID STEM CELL
ACUTE LYMPHOBLASTIC LEUKEMIAS
PRECURSOR T - CELL
PRECURSOR B - CELL
LYMPHOBLASTIC LYMPHOMAS / LEUKEMIAS
MATURE T - CELL
MATURE B - CELL
NON-HODGKINS LYMPHOMAS
S. J. Schuster, 2003
LYMPH NODES, EXTRANODAL TISSUES
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Chronic lymphocytic leukemia
LEUKEMIA

• A Group of neoplastic diseases of the leukocyte
• Cancer of leukocytes or their precursors.
• Accumulation or proliferation of leukocytes in
  the bone marrow.
• May or may not have increased leukocyte count in
  the bone marrow.

• Is characterised by the accumulation of
  non-proliferating mature-appearing lymphocytes in
  the blood, marrow, lymph nodes, and spleen
• Is the most common form of leukemia in North
  America and Europe, but is extremely rare in the
  Orient

• Commonest leukemia in adults
• Typically occurs in older patients, with the
  highest incidence being in those aged 50 to 55
  years
• Affects men twice as often as women
• Does not affect children
• Approximately 25 of all leukemias

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Normal Blood
Reticulocyte
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Lymphocytes in normal case
lymphocytes
CLL - blood count
Lymphocytes in case of CLL
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CML - pathology
Chronic Myeloid Leukemia
A neoplasm of hematopoietic stem cells A
two-phase disease
CML - clinical features of chronic phase

• Peak age 20 to 40 years ,Weight loss, night
  sweats ,Big spleen ,Gout ,Often found by chance

• Chronic Phase
• Accumulation of myeloid cells
• bone marrow
• peripheral blood
• spleen and liver

• Accelerated Phase
• Further genetic changes in the stem cell leading
  eventually to acute transformation (ie acute
  leukemia)

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CML - blood count
neutrophil
basophil
myelocytes
metamyelocyte
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LYPHOMA
Any Malignant tumour, or condition allied to a
tumour , arising from Some or all of the cells of
lymphoid tissue.

• Lymphoma (Hodgkins and non-Hodgkins) is the
  third most common childhood malignancy.
• Non-Hodgkins lymphoma (NHL) accounts for
  approximately 7 of cancers in children less than
  20 years of age.
• In the United States, there are about 800 new
  cases of NHL diagnosed each year.
• Incidence is approximately 10 per 1,000,000.

• Although there is no sharp age peak, occurs most
  commonly in the second decade of life, and occurs
  less frequently in children less than 3 years of
  age.
• NHL is the most frequent malignancy in children
  with AIDS, and it often occurs before the age of
  4 years in those who have vertical transmission
  of the virus.
• Screening for HIV should be considered for all
  children with NHL.

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Cellular Classification
LYPHOMA

• In children, non-Hodgkins lymphomas (NHLs) are
  distinct from the more common forms of lymphomas
  in adults.
• While lymphomas in adults are more commonly of
  low or intermediate grade, almost all that occur
  in children are high grade.

• NHL in children can be classified into 1 of 4
  categories
• Burkitts and Burkitts-like lymphoma/small
  noncleaved B cell lymphoma
• Lymphoblastic lymphoma
• Diffuse large cell lymphoma.
• Anaplastic large cell lymphoma

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Lymphoblastic Lymphoma

• Involvement of the bone marrow may lead to
  confusion as to whether the patient has lymphoma
  or leukemia.
• Traditionally, patients with greater than 25
  marrow blasts are considered to have leukemia,
  and those with less than 25 marrow blasts are
  considered to have lymphoma.

• Lymphoblastic lymphomas are usually positive for
  the enzyme terminal deoxynucleotidyl transferase
  (TDT) and have a T-cell immunophenotype.
• About 10 to 15 of lymphoblastic lymphomas have
  non-T immunologic characteristics (for example,
  common acute lymphocytic leukemia antigen
  CD10-positive precursor B-cell phenotype).
• Chromosomal abnormalities are not well
  characterized in patients with lymphoblastic
  lymphoma.

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Lymphoblastic Lymphoma

• Makes up approximately 30 of childhood NHL.
• Predominantly tumors of thymocyte (T-cell)
  origin.
• 75 of patients with lymphoblastic lymphoma have
  an anterior mediastinal mass, and may present
  with symptoms of dyspnea, wheezing, stridor,
  dysphygia, or swelling of the head and neck.
• Pleural effusions may be present.
• Involvement of lymph nodes, usually above the
  diaphragm, may be a prominent feature.

• There may also be involvement of bone, skin, bone
  marrow, central nervous system, abdominal organs
  (but rarely bowel), and occasionally other sites
  such as lymphoid tissue of testis.
• Abdominal involvement is rare most patients with
  an abdominal mass have small noncleaved cell or
  large cell lymphoma.
• Localized lymphoblastic lymphoma may occur in
  lymph nodes, bone, subcutaneous tissue, etc.

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Large Cell Lymphoma

• This is heterogeneous group of tumors accounting
  for approximately 20 to 25 of childhood NHL.
• While the pathological types of LCL as described
  in REAL and WHO classification are anaplastic
  large cell and diffuse large cell, treatment
  decisions in pediatric LCLs are based on
  immunophenotype.

• Biologically, B-cell LCL occasionally is similar
  to small noncleaved cell lymphoma both with
  respect to immunophenotyping and chromosomal
  abnormalities (e.g., presence of an
  (814)translocation).

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Large Cell Lymphoma

• T-lineage LCL can be divided into CD30
  (Ki-1)-positive anaplastic LCL and other
  peripheral T-cell lymphoma.
• While the predominant immunophenotype of the
  anaplastic LCLs is T-cell, null-cell types have
  also been described.
• More than 90 of anaplastic LCLs are
  CD30-positive and have the nonrandom
  translocation (25), (p23q35) leading to the
  expression of the fusion protein NPM ALK.

• Clinically, anaplastic LCL has a broad range of
  presentations, including involvement of lymph
  nodes and a variety of extranodal sites,
  particularly skin, bone, and less often
  gastrointestinal tract, lung, pleura, and muscle.
• Involvement of the CNS and bone marrow is
  uncommon.
• These are often associated with systemic symptoms
  (e.g., fever, weight loss) and a prolonged waxing
  and waning course.

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Small Noncleaved Cell Lymphoma

• Burkitts and Burkitts-like.
• Accounts for 40 to 50 of childhood NHL and
  exhibits consistent clinical behavior.
• Up to 90 of these tumors are intra-abdominal.
• Other sites testis, nasal sinuses, bone,
  peripheral lymph nodes, skin, bone marrow, and
  CNS.
• Small noncleaved cell lymphoma are of B-cell
  origin they usually express surface
  immunoglobulin M (IgM) of either Kappa or Lambda
  light chain subtype.

• These tumors also express a characteristic
  chromosomal translocation, usually t(814) and
  more rarely t(822) or t(28).
• Each of these translocations juxtaposes the c-myc
  gene to immunoglobulin locus regulatory elements,
  resulting in the inappropriate expression of
  c-myc, a gene involved in cellular proliferation.
  

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NON HODGKIN LYMPHOMA
CLINICAL GROUPS

• Indolent
• generally slow growing untreated survival
  measured in years.
• common types include follicular, small
  lymphocytic, marginal zone, cutaneous T-cell
  lymphoma, Waldenstroms.
• Aggressive
• grow quickly untreated survival measured in
  months.
• common types include diffuse large B-cell,
  mantle cell, and peripheral T-cell lymphoma.
• Highly Aggressive
• extremely rapid growth untreated survival
  measured in weeks.
• common types include Burkitts and
  lymphoblastic.

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NON HODGKIN LYMPHOMA

• Possible causes
• Individuals exposed to chemicals (pesticides,
  fertilizers or solvents)
• Individuals with compromised immune systems
• Heredity
• Certain individuals infected with human
  T-lymphotropic virus type I or HIV (AIDS)
• Most patients have no clear risk factors
• Exact cause is unknown

• 53,400 people in the US are expected to be newly
  diagnosed with NHL this year1
• Approximately 300,000 people are currently
  living with the disease2
• American Cancer Society, Cancer Facts and Figures
  2003
• SEER Cancer Statistics 1973-1999

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THROMBOCYTOPENIA
An abnormal decrease or deficiency of leukocytes
circulating in the blood

• Risk of bleeding
• platelet count
• cause of thrombocytopenia
• comorbid disease
• drugs

• Clinical manifestations
• petechiae
• purpura, ecchymoses
• mucosal bleeding
• menorrhagia
• intracranial bleeding

formation of demarcation membranes
platelets
megakaryocyte
(pro)platelets
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THROMBOCYTOPENIA
Leukocytes (x 109/L) 6.8 4.0 -
11.0 Hemoglobin (g/L) 130 120-160 MCV (fL)
87 80 - 100 Platelet count (x
109/L) 11 150 - 450 MPV
(fL) 12.5 7.4 -10.4
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CD40 receptor CD40 L (CD154)
CD30 receptors CD30 L(CD153)
MEMBERS OF TNF SUPERFAMILY
RANK RANKL
TRAIL (Apo2L) its receptors
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CD30 RECEPTOR

• CD30 is a 120-kilodalton (kD), type I
  transmembrane protein that contains 6
  cysteine-rich pseudorepeat motifs in its
  extracellular domain .
• In healthy individuals, CD30 expression is
  restricted to a small number of activated B and T
  lymphocytes.
• Like many of the TNF receptors, CD30 can be shed
  in a soluble form (sCD30). An 85 kD sCD30 can be
  detected in culture supernatants of CD30 cell
  lines and sera of patients having CD30 tumors.
• sCD30 is detected at low levels in the sera of
  healthy individuals and in individuals who are
  infected with one of several different viruses,
  including hepatitis B and C, HIV, and EBV, or
  have an autoimmune disorder.
• An elevated level of serum sCD30 expression in
  patients with anaplastic large-cell lymphoma or
  Hodgkin disease is correlated with a poor
  prognosis, perhaps because a high level of sCD30
  reflects a high tumor burden or because sCD30L
  may block the biologic effects of CD30L.

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Hodgkin disease
infectious mononucleosis
lymphomatoid papulosis
immunoblastic lymphoma
CD30 EXPRESSION
adult T-cell lymphoma leukemia

• .
• activated B and T lymphocytes

anaplastic large-cell lymphoma
multiple myeloma,
mycosis fungoides
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CD30L

• CD30L (CD153) is a type II transmembrane protein
  that belongs to the TNFSF.
• The human CD30L gene has been mapped to
  chromosome 9q33
• Like many TNF receptors, CD30s cytoplasmic tail
  contains several TRAF-binding sequences that can
  bind TRAF-1, -2, -3, and -5. Both TRAF-2 and
  TRAF-5 have been implicated in CD30 activation of
  NF-?B

• Resting B cell
• activated B cells
• activated T cells,
• natural killer (NK) cells
• Eosinophils
• granulocytes,
• monocytes,
• mast cells.
• epithelial cells
• Hassall corpuscles in the thymus medulla.

• chronic lymphocytic leukemia (CLL),
• follicular B-cell lymphoma,
• hairy cell leukemia,
• T-cell lymphoblastic
• lymphoma, and
• adult T-cell leukemia
• lymphoma.
• Cells infected with

CD30 L EXPRESSION
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CD30L

• PHYSIOLOGIC FUNCTIONS
• Role in T-cell costimulation
• Cytokine and chemokine secretion,
• Regulation of class-switch DNA recombination,
• Antibody production in subsets of human Bcells
• CD30L has diverse biologic activity in different
  CD30 cancers, ranging from enhancement of
  survival to no induction of apoptosis and cell
  cycle arre
• These paradoxical effects are caused by a
  differential effect on NF-B activation..

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CD40

• CD40 is a 50 kD,
• Type I transmembrane protein
• Extracellular segment containing 4 cysteine-rich
  repeats
• Its intracellular tail has no DD or kinase
  activity
• It can activate several second messenger
  pathways, including MAP kinases (ERK, c-jun amino
  terminal kinase JNK, and p38) and NF-B. This
  activation is mediated by interacting with
  several TRAFs, including TRAF-2,-3,-5, and -6.

• CD 30 EXPRESSION
• Non hematopoietic cells urinary bladder, ovary,
  breast, and liver.
• Hematopoietic cells normal B lymphocytes,
  monocytes, and dendritic cells and subset of T
  cells.
• Malignant cells B- and T-cell lymphomas and H/RS
  cells in Hodgkins disease
• Endothelial cells and EBV-associated
  nasopharyngeal carcinoma. Expressed more
  frequently and intensely in mature B lymphocytes
  than in precursor lymphoblastic leukemia cells

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CD40L

• CD 40 L EXPRESSION
• Activated T lymphocytes (more frequently in CD4
  than in CD8 lymphocytes)
• Activated B lymphocytes, NK cells, monocytes,
  basophils, eosinophils, dendritic cells, and
  platelets
• Nonhematopoietic cells, including endothelial and
  smooth muscle cells.
• In humans, constitutive expression of CD40L has
  been observed in T cells of patients having
  autoimmune disorders and in malignant B and
  Tlymphocytes of patients with lymphoma or
  leukemia.

• 3233 kDa.
• Type II transmembrane protein.
• Also known as CD154
• The human CD40L gene is located on chromosome
  X26.327.1.
• It also can be shed into smaller biologically
  active soluble forms, which can be detected in
  sera of patients with lymphoid malignancies,
  autoimmune disease, or essential thrombocythemia,
  but not in sera of healthy individuals.
• Physiologic functions priming dendritic cells to
  activate CD8 cytotoxic T cells, activation,
  survival, proliferation, differentiation, and
  immunoglobulin isotype switching of B cells,
  enhancing antigen presentation by means of
  up-regulation of their CD80 (B7.1) and CD86
  (B7.2) costimulatory molecules, and regulating
  cytokine and chemokine secretion

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RANKL/OPGL/TRANCE/ODF

• RANKL, also known as osteoprotegerin (OPG)
  ligand, TRANCE, and osteoclast differentiation
  factor (ODF).
• 317amino acid.
• Type II transmembrane protein that belongs to the
  TNFSF.
• Has highest sequence homology to CD40L and TRAIL
  .
• In addition to the 4045 kD transmembrane form,
  RANKL also exists in a smaller, 31 kD soluble
  form.
• RANKL has two receptors a transmembrane protein,
  called RANK, and a soluble protein dimer, called
  OPG.
• RANK is expressed primarily in activated T cells
  and osteoblasts. .
• Recent data have suggested that the RANKL/RANK
  system may be involved in the pathogenesis of
  human hematologic disorders, including Hodgkin
  disease and multiple myeloma.

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TRAIL (Apo2L)

• TRAIL is a type II membrane protein that shares
  the highest sequence homology in its
  extracellular domain with FasL (28) and TNF-
  (23).
• The human TRAIL gene has been mapped to
  chromosome 3q26 . .
• TRAIL has four exclusive receptors DR4, DR5,
  DcR1, and DcR2 .
• The genes for these four receptors are clustered
  on the short arm of chromosome 8 (8p21).
• DR4 (TRAIL-R1)and DR5 (TRAIL-R2, Killer, TRICK2)
  are death receptors that contain a DD in their
  intracellular tail. Both receptors recruit a
  Fas-associated DD adaptor protein and activate
  caspase-8 and caspase-10.
• In addition to activating death pathways, TRAIL
  can also activate NF-B and JNK9396 and the
  nitric oxide pathway. The two remaining receptors
  are decoy receptors that do not transduce
  apoptotic signals. Specifically, DcR1 (TRAIL-R3,
  TRID, LIT) lacks an intracellular tail, whereas
  DcR2 (TRAIL-R4, TRUNDD) has an incomplete DD in
  its intracellular tail.

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TRAIL RECEPTOR
Membrane
Death domain
DECOY RECEPTOR
DEATH RECEPTOR
Decoy receptors Bind ligand, but not able to
signal
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TRAIL is a ligand of particular importance
TRAIL's death receptors (TRAIL-R1 and TRAIL-R2)
are mainly expressed in transformed cells
TRAILs decoy receptors (TRAIL-R3, TRAIL-R4 and
TRAIL-R5) are expressed in normal cells. 
TRAIL kill a wide variety of tumor cells with
minimal effects on normal cells.
Unfortunately, TRAIL is very toxic to human
hepatocytes.
But harmless for mouse hepatocytes (mice is
wrong model again)
Tat protein, released from HIV-infected cells,
can induce production of TRAIL by macrophages,
resulting in the apoptosis of bystander T cells 
TRAIL in AIDS
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TRAIL PHYSIOLOGICAL FUNCTION

• TRAIL has protective antitumor function mediated
  by TRAIL expressed by NK cells.
• TRAIL may also play a role in regulating cytokine
  and chemokine expression.
• TRAIL has a wide range of activity against human
  primary tumor cells and cell lines.
• In tumors of hematologic origin, TRAILs
  strongest killing activity is observed in Jurkat
  (T-cell lymphoblastic lymphoma/ leukemia) and
  8226 (multiple myeloma) cell lines, In these cell
  lines, the activity of TRAIL was comparable with
  that of FasL

• TRAIL induced a moderate cell proliferation
  rather than cell death in 8226 (multiple myeloma)
  cell lines.
• TRAIL has been shown to be effective in primary
  and cultured multiple myeloma cells, including
  those that are resistant to doxorubicin
• TRAIL activates both the death receptor pathway
  and mitochondria pathway, as evidenced by the
  rapid activation of caspase-8, -9, and -3.

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TRAIL EXPRESSION

• Primary CLL.
• Chronic and acute leukemia.
• TRAIL induced cell death in a variety of AML cell
  lines.
• Primary acute lymphoblastic leukemia, acute
  myeloid leukemia, CLL, multiple myeloma, and
  chronic myelogenous leukemia cells.

• Tumor cells expressed functional TRAIL that
  killed target Jurkat cells by means of a
  TRAIL-specific mechanism.
• In multiple myeloma cells implicated in the
  pathogenesis of anemia associated with multiple
  myeloma.

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