Resistance to Allogeneic Bone Marrow Transplantation

1
Resistance to Allogeneic Bone Marrow
Transplantation

• Claudio Mosse MD, PhD
• Johns Hopkins Hospital
• Department of Pathology

2
Goal Use the Immune System to Cure Tumors

• Tumor vaccines to prime the host immune system
  against host tumor
• Tumor specific antigens vs tumor associated
  antigens
• Overcoming tolerance
• Weakened host hematopoiesis
• Transplant a new immune system
• Graft versus Host Disease (GvHD)
• Infections

3
Indications for Allogeneic Bone Marrow
Transplantation

• Stem cell source to correct genetic diseases
• Hemoglobinopathies
• Sickle cell anemia
• Thalassemia Major
• Metabolic/Enzymatic deficiencies
• Severe Combined Immunodeficiency
• Mucopolysaccharidoses

4
Indications for Allogeneic Bone Marrow
Transplantation

• Immunotherapy for malignancy
• Solid tumors
• Breast, renal, colon, ovarian and sarcomas
• Hematopoietic tumors
• CML, Follicular lymphoma
• Aggressive B cell lymphomas
• Hodgkin lymphoma
• Acute myeloid leukemia, acute lymphoblastic
  leukemia/lymphoma
• Myelodysplastic syndrome

5
Indications for Blood and Marrow Transplantation
in North America 2002
4,500
Allogeneic (Total N 7,200) Autologous (Total N
10,500)
4,000
3,500
3,000
2,500
TRANSPLANTS
2,000
1,500
1,000
500
0
NHL
MultipleMyeloma
AML
CML
MDS / OtherLeukemia
Neuroblastoma
Non-MalignantDisease
6
Evidence for an Immunotherapeutic Effect
CML

• Twin (syngeneic) vs. allogeneic transplant has
  higher rate of relapse.

Gale RP et. al. Ann Intern Med 1994120646-652
7
Allogeneic BMT Cures Leukemia/ Lymphoma
Horowitz MM et al, Blood. 1990 Feb
175(3)555-62.
8
Further Evidence for an Immunotherapeutic Effect

• Relapse after treatment can be treated with Donor
  Lymphocyte Infusion (DLI) that causes mild graft
  versus host disease (GvHD). Helg C et al, Leuk
  Lymphoma. 1998 Apr29(3-4)301-13.
• Relapse is more common in patients with mixed
  chimerism than in those with complete conversion.
  Bader P et al, Bone Marrow Transplantation, 1998,
  21 487-95.

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Causes of Death After Transplantation (1996-2000)
www.ibmtr.org
10
Summary

• AlloBMT is successful because of the associated
  allogeneic immune response evidenced by GvHD.
• GvHD kills roughly 15 of patients with
  allogeneic bone marrow transplantation.
• Is there a way to decrease GvHD without
  compromising GvL?

11
Nonmyeloablative Bone Marrow Transplants - aka
miniBMT

• Less stringent conditioning and fewer
  transplanted CD34 cells leads to mixed chimerism
  between host and donor.
• Decreases window of neutropenia, lessening
  infectious complications Slavin S et al, Blood.
  1998 Feb 191(3)756-63.
• Decreases frequency and severity of GvHD without
  significant sacrifice of GvL. Mielcarek M et al,
  Blood. 2003 Jul 15102(2)756-62.

12
Decreased aGvHD in miniBMT particularly miniMUDs
Sibling
MUD
Mielcarek M et al, Blood. 2003 Jul
15102(2)756-62.
13
Donor Lymphocyte Infusion

• DLI boost of donor mature T cells that
  trigger a bout of acute GvHD that may kill off
  recurring tumor (GvL effect). Spitzer TM et al,
  Biol Blood Marrow Transplant. 20006(3A)309-20.

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Summary

• Transplanted immune system is not tolerized.
• Graft versus host disease an allogeneic immune
  reaction of donor marrow against host tissues
  needed for tumor rejection.
• Host hematopoietic cells are recognized by the
  transplanted immune system.
• Patients rarely maintain host hematopoiesis even
  in nonmyeloablative transplantation, typically
  converting to complete donor hematopoiesis after
  DLI.

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Causes of Death After Transplantation (1996-2000)
www.ibmtr.org
16
Paradox

• Why does the host hematopoietic tumor continue to
  grow in the face of GVHD and full donor
  chimerism?
• Loss of MHC only on tumors
• NK cells should lyse MHC negative cell lines
• Published data Kluin PM and Grogan TM
• Inability to die
• Loss of apoptosis pathway members

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Apoptosis

• Programmed cell death
• Requires energy
• Ordered dismantling of cell structure
• Mitochondrial outer membrane permeabilization
• Protein phosphorylation and synthesis
• DNA laddering
• Plasma membrane blebbing
• Phagocytosis of cellular packets by MF

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Extrinsic Pathway

• Death receptor or Fas trimerization on binding
  their respective ligands leads to activation of
  caspases 8 and 10 followed by caspases 3, 6 and
  7.
• Decoy receptors exist that bind TRAIL without
  transducing a death signal
• FLIP inhibits caspase 8 activation
• Granzyme B can activate all of these caspases
  directly

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TRAIL
FasL
Extrinsic Pathway Of Apoptosis
DR
Fas
FADD
FLIP
Casp 8
Gran B
Casp 3
Casp 7
DNA Fragmentation Chromatin condensation
Casp 6
Membrane blebbing
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Intrinsic Pathway

• Cellular stresses (DNA damage, cytokine
  deprivation) change ratio of pro-apoptotic to
  anti-apoptotic bcl-2 family members
• Mitochondrial outer membrane permeabilized by
  dimers of pro-apoptotic members
• Apaf-1/ Cytochrome c activate caspase 9
• Smac inhibits IAPs that block caspase 9

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Intrinsic Pathway Of Apoptosis
Survival factors, Growth factors, cytokines
Bcl-xL, Bcl-2
Bad
Bax, Bak
Bax, Bak
p53
DNA damage
Bax
Bax
Casp 8
Bid
Apaf-1
Cyto c
Casp 9
Extrinsic pathway
Casp 3
Apoptosis
Casp 7
IAPs
SMAC
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Hypothesis

• Dysregulation of apoptosis pathways can protect
  leukemias and lymphomas from cytotoxic cell
  induced apoptosis.

23
Predictions

• Anti-apoptotic proteins are over-expressed, and
  pro-apoptotic proteins are under-expressed in
  leukemias and lymphomas.
• The tumors with these changes are more
  aggressive/ have a worse prognosis.
• Reversing these changes should restore
  sensitivity to cytotoxic cell induced apoptosis.

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1. Dysregulation of Apoptosis Family Members in
Leukemia and Lymphoma

• Decreased Fas cHL, ALL, CLL, CML, FL, BL,
  DLBCL, HCL
• Decreased Fas signaling NK/TcL
• Increased FLIP cHL
• Increased bcl-2 FL, ALL, CLL, CML, HL, MALT,
• Increased bcl-xL MM, FL
• Increased IAPs DLBCL, ALCL, MCL

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2. Tumors with Apoptosis Pathway Defects Have a
Worse Prognosis.

• In vitro bcl-2 over-expression inhibits Fas- and
  TRAIL-mediated apoptosis.
• Increased bcl-2, poor prognostic factor in DLBCL,
  and associated with chemotherapy resistance in
  CLL, DLBCL, ALCL, AML and HL.
• Increased bcl-xL is a poor prognostic factor in
  FL.

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3. Reversing These Changes Will Restore
Apoptosis Sensitivity

• Bortezimib - proteasomal/ NF-kB inhibitor
• Genasense - bcl-2 antioligonucleotide
• Rapamycin - Akt inhibition

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Bcl-xL

• Bcl-2 family member anti-apoptotic
• Stabilizes mitochondrial outer membrane
• Inhibits bax, bak and other pro-apoptotic bcl-2
  family members
• Increased levels as poor prognostic factor in FL
• Does it play a role in resistance to T cell
  induced apoptosis in DLBCL?

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Creation of a Bcl-xL Vector
retroviral vector pMX/IRES-GFP transfected into
BOSC3 packaging cell line
Bcl-xL
5 LTR
3 LTR
IRES GFP
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Fas and TRAIL-induced apoptosis
anti-Fas Ab (1ug/ml) or TRAIL (1ug/ml)
20 hr
200
160
TRAIL
no treatment
120
80
Fas
40
Annexin V
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Bcl-xL Inhibits In Vitro Fas-induced, but not
TRAIL-induced Apoptosis
31
Bortezomib (PS-341)

• Dipeptidyl boron compound
• Proteasome inhibitor
• Approved for multiple myeloma in patients with 2
  previous treatment failures
• Investigational drug in wide range of lymphomas,
  leukemias and solid tumors

32
Mitsiades, Nicholas et al. (2002) Proc. Natl.
Acad. Sci. USA 99, 14374-14379
33
PS-341 (Velcade) Synergizes with Fas to induce
Apoptosis
34
PS-341 Synergizes with TRAIL to induce Apoptosis
35
Allogeneic CTL assay
hn
hn
d6
d8

3H-Thy
36
Bcl-xL Does Not Inhibit Allogeneic T Cell-Induced
Apoptosis
37
Allogeneic Bone Marrow Transplantation In Vivo
Model
Balb/c
Day 0 TUMOR 1 x 106 A20 or R2 BMT 1 x 107
B10.D2 BM IV DLI 0, 1 x 106, 1 x 107, or 5 x
107 B10.D2 splenocytes IV
Day 1 850 cGy TBI
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Bcl-xL over-expression does not protect A20 from
alloBMT and DLI
A20, 0 DLI
A20, 1 x 106 DLI
A20, 10 x 106 DLI
A20, 50 x 106 DLI
P0.001
bcl-xL, 0 DLI
bcl-xL, 1 x 106 DLI
bcl-xL, 10 x 106 DLI
bcl-xL, 50 x 106 DLI
39
Conclusions

• Bcl-xL over-expression in A20 can confer
  resistance to Fas-induced apoptosis in vitro.
• TRAIL-induced apoptosis is not affected by bcl-xL
  over-expression.
• Bcl-xL over-expression in A20 does not protect in
  vivo from alloBMT and DLI.
• Discordance between in vitro and in vivo data
  Fas inhibition does not affect overall protection
  from alloBMT and DLI.

40
Future Plans

• Systematically test apoptosis pathway members for
  ability to confer Fas, TRAIL, alloCTL and alloBMT
  resistance.
• Using IHC and IF for apoptosis family members,
  examine human lymphomas from patients treated
  with alloBMT and correlating with outcome.
• Confirm findings from human specimens using A20
  model in mice.
• Reverse apoptosis resistance using targeted
  therapeutics.

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1. Screen Apoptosis Members

• Transfect in genes for anti-apoptotic proteins or
  siRNAs for pro-apoptotic proteins.
• Use previously published dysregulated proteins
• Systematically work from distal to proximal on
  pathway

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1. Screen Apoptosis Members

• Compare expression of apoptosis pathway genes
  from alloBMT-resistant cell lines with
  alloBMT-sensitive cell lines.
• Existing human lines from relapse and cured
  patients
• Solid tumors vs hematopoietic tumors
• Develop in vitro and in vivo selection techniques
  to generate alloBMT-resistant clones from
  alloBMT-sensitive clones.
• In vitro alloCTL as selection agents
• In vivo alloBMT model with A20 suboptimally
  treated and harvested

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2. Examine Patient Specimens for Correlation
between Apoptosis Protein Expression and Survival

• Roughly 50 DLBCLs half cured and half with
  relapse will be screened for multiple apoptosis
  pathway members.
• Use tissue microarrays to rapidly screen multiple
  specimens at a time on one slide with homogenous
  staining conditions
• Chromavision technology to quantify staining
  intensity

44
Slide from Mark A. Rubin, M.D., U. Michigan
45
2. Examine Patient Specimens for Correlation
between Apoptosis Protein Expression and Survival

• Start distally and work proximally TUNEL to
  Fas, DR, bcl-2 family members
• Correlate with chemosensitivity, alloBMT
  sensitivity, survival.

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3. Test Patient Findings in Murine Model

• Transfect gene or siRNA into A20 and test with in
  vitro Fas and TRAIL induced apoptosis.
• Use allogeneic cytotoxic T cells in an in vitro
  assay to measure change in sensitivity to
  apoptosis.
• Use in vivo model to examine resistance to
  alloBMT.

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4. Use Targeted Therapeutics to Correct
Apoptosis Defect

• Use treated tumor in Fas and TRAIL assays.
• Use treated tumor in allogeneic CTL assay.
• AlloBMT and DLI combined with inhibitor in mice
  bearing tumor.

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4. Use Targeted Therapeutics to Correct
Apoptosis Defect
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So what

• Role of the pathologist is to study disease.
• to define and diagnose disease.
• to work in concert with clinicians to best
  direct patient care and management.
• Goal of the tumor immunologist is to use the
  immune stystem to eradicate tumors.
• Identify targets and increase tumor cell
  susceptibility to the immune system.

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Acknowledgements

• Ephraim Fuchs, MD
• Sanju Jalla, MS
• Jie Wang, MD
• Michael Borowitz, MD/PhD
• Frederick Racke, MD/PhD
• Angelo DeMarzo, MD/PhD
• Lymphoma SPORE grant