Michael Tomasson, MD HematologyOncology Grand Rounds 111403

1
Michael Tomasson, MDHematology/OncologyGrand
Rounds11/14/03
Development of an animal model of t(414)
multiple myeloma
2
Requirements for malignant transformation
Self-sufficiency in growth signals
Limitless replicative potential
Hanahan Weinberg, Cell 2000
3
Requirements for a career in academia
Get up go
Thrive on criticism
Dont quit
Obtain funding
Local invasion or metastasis
Be obsessive
4
Recurring chromosomal translocations in human
leukemia
5
Requirements for malignant transformation
Hanahan Weinberg, Cell 2000
6
Recurring chromosomal translocations in multiple
myeloma
7
t(4 14) translocation dysregulation of FGFR3
and MMSET
FGFR3
4p16.3
MMSET
bkpt
14
4
Cen.
Tel.
der(4)
der(14)
8
FGFR3 an RTK involved in MM pathogenesis

• One of four tyrosine kinase receptors of FGF
  family
• Germline mutations that cause FGFR3 activated in
  the absence of ligand are responsible for
  inhibition of normal bone growth
• Normal B cells do not express FGFR3
• Elevated FGFR3 level found in MM cells with
  t(414) translocation
• Mutated FGFR3 can transform hematopoietic cells,
  but not MM, WT FGFR3 need longer latency (gt1 yr)

9
Clinical facts about Multiple Myeloma

• Malignant B cell tumor -- multi-focal tumors in
  bone marrow
• Clongenic plasma cells secrete immunoglobulins
  (Ig) or Ig fragments light chain deposition
  disease is a variant
• Progression from MGUS to MM
• Pathophysiologic features include osteolytic
  lesions, anemia, renal insufficiency and
  recurrent bacterial infection
• Incurable 3-4 year survival with routine therapy
• 4-5 year survival with high-dose therapy

10
Stromal cell interactions appear to be important
to MM pathogenesis
11
Microarrays have been used extensively to study MM
12
HD Rx in Multiple Myeloma
13
Recurring chromosomal translocations in multiple
myeloma
14
Multi-step pathogenesis of MM
a Normal long-lived Plasma cell
? 13
Germinal-centre B cell
Myeloma Cell line
Extramedulary Myeloma
Intramedulary Myeloma
Smouldering Myeloma
MGUS
Bone marrow stromal cell dependences
IL-6 dependences
Angiogenesis
Bone destruction
Increased DNA-labeling index
b c d
e
15
FGFR3 is overexpressed in most, but not all
t(414)
16
Is MMSET critical for MM transformation?

• MMSET mRNA is expressed in all human multiple
  myeloma cell lines
• Significantly increased level of MMSET in MM
  cells with t(414) translocation
• t(414) translocation is related to poor
  prognosis
• MMSET expression may be independent of FGFR3 due
  to loss of der(14)
• Importantly no examples of t(414) tumors have
  lost der(4) or do not contain Ig/MMSET hybrid
  transcripts
• High expression of MMSET is correspondent to
  shorter survival
• Strikingly, share significant homology with
  HRX/ALL-1/MLL gene
• No protein study has been reported

17
MMSET (Multiple Myeloma SET domain)

• Same as WHSC1 (Wolf-Hirshhorn Syndrome)
• SET (Suppressor of variegation-Enhancer of
  zeste-Trithorax)
• Other functional domains suggest nuclear
  localization/DNA binding
• Expressed in normal tissue thymus and testes
• Two spliced mRNA

8389 PA
Polydenylation Signals
ORF 1911 bp
5UT
2092
2990
3095
3266
8364

3
11
4
5
6
7
8
9
10
NLS
NLS
PHD
PHD Finger

hath
HMG
1
2
3
hath
SET
4
7418 PA
ORF 4094 bp
PA Signals
5UT
4982
7395

24
3
4
5
6
7
8
9
10
12
13
14
15
16
17
18
19
20 21 22 23
18
Anti-MMSET Serum
L S G C C
L
S
G
C

• Synthesize peptide KQSPLSVQS
• Immune rabbits
• Purify Serum
• Immunoblot with lysate from MMSET cDNA
  transfected BaF3 cells

110
61
Control
GFP
Short
Long
110
61
36
19
First step - Retrovirus in vitro and in vivo
MSCV-MMSET-long-GFP MSCV-MMSET-Short-GFP MSCV-GFP
MSCV-MMSET-long-Neo MSCV-MMSET-Short-Neo MSCV-Neo
LTR
MMSET
IRES
eGFP
LTR
20
(No Transcript)
21
Retroviral expression in IL-3 dependent BaF3
cells

• MMSET could not rescue IL-3 starved BaF3 cells

22
Primary murine bone marrow transduction/transplant
ation
IL-3, SCF, TPO, FLT-3
5-FU
Total BM
Transduce with virus
Harvest transduced cells
23
MMSET BMT Spleen
(Control)
(GFP)
0.00
0.06
11.20
60.43
16.41
80.39
(MMSET-Long)
(MMSET-Short)
1.22
28.59
0.89
17.52
GFP
63.13
56.87
B220
24
Spleen
(Control)
(GFP)
21.43
32.34
0.02
0.23
20.53
44.22
(MMSET-Long)
(MMSET-Short)
5.55
4.76
11.03
5.98
GFP
33.19
32.37
B220
25
Peripheral Blood Analysis
26
Another approach Transgenic mice

• 5 E-mu-MMSET (MMSET-long and MMSET-short)
• Target expression of MMSET to B cells using
  well-studied immunoglobulin heavy chain enhancer
  and promoter casette

Eµ VHP
MMSET
ß-globin splice Poly A
3 MMSET-Long transgenic founders4 MMSET-Short
transgenic founders
27
MMSET RT-PCR of transgenic mice
3T3 Long
TG Spleen
TG B.M.
BMT Spleen
BMT B.M.
GFP Spleen
GFP B.M.
28
MM is a cancer of post-GC B-cells
29
Ongoing MM experiments

• Develop MMSET antisera
• Test hypothesis that MMSETL and S isoforms
  heterodimerize
• Sheep RBC stimulate MMSET mice assess post-GC
  B-cells
• Cross MMSET and Rb/- mice
• GC-cre x activated Ras ( x MMSET)
• MMSET knock-out?

30
DJ
3E?
P
P
E?
I?
Chr. 14q32
?
P
P
P
1
FGFR3
3
MMSET
Chr. 4p16
t(414)
CENTROMERE
TELOMERE
p
P
P
E?
MMSET
3
der(4)
I?
3E?
P
P
1
FGFR3
?
der(14)
Fig. 2 Dysregulation of FGFR3 and MMSET/WHSC1 by
t(414)(p16q32) in MM
Bergsagel PL, Oncogene, 2001
31
Whitlock-Witte long-term B-cell Culture

• Harvest BM cells from Balb/c mice
• Retrovirus infection
• Culture in RPMI 1640 5FBS 50 ?M 2-ME, and
  change media twice a week
• Count floating cells after 16 d and every 3 days
  thereafter

32
Isotype Control
GFP
84.92
9.78
12.16
9.62
MMSET-Long
MMSET-Short
85.04
10.41
79.23
9.43
B220
GFP
33
Retrovirus infected BMT

• Donor 5-Fu treated Balb/c mice (n10 per
  construct)
• Recipient 900 rad irradiated Balb/c mice (n10)
• So far, Mice are happy after 11 weeks

34
MMSET BMT Spleen
0.43
71.55
1.83
26.92
15.98
0.44
GFP
1.48
4.11
4.53
GFP
MMSET-Long
MMSET-Short
CD3
35
Bone Marrow
37.97
3.98
49.52
1.58
64.49
21.00
32.93
26.21
GFP
Gr
GFP
MMSET-Long
MMSET-Short
36
Bone Marrow
28.31
56.00
2.09
20.60
1.49
20.83
9.16
60.40
60.88
GFP
MMSET-Long
GFP
MMSET-Short
Mac
37
(GFP)
51.45
17.87
Spleen
10.34
(MMSET-Long)
17.02
4.43
36.68
(MMSET-Short)
10.02
2.97
GFP
26.02
CD3
38
(GFP)
54.92
1.68
Spleen
0.79
(MMSET-Long)
17.64
0.96
1.61
(MMSET-Short)
1.34
11.80
GFP
6.63
Gr
39
(GFP)
54.92
1.68
Spleen
0.79
(MMSET-Long)
17.64
0.96
1.61
(MMSET-Short)
11.80
1.34
GFP
6.63
Mac
40
MMSET domains

• NLS Nuclear Localization Signal
• HMG box High Mobility Group
• hath motif - homologous to the amino terminus of
  hepatoma derived groth factor HDGF-has an NLS
  and HMG
• PHD - plant homeodomain zinc finger
• SET - Suppressor of variegation-Enhancer of
  zeste-Trithorax

41
FGFR3
MMSET
3E ? ? ? ? ? E? VDJ
3E
E?
14
4
42
(No Transcript)
43
Summary of Multiple Myeloma

• Fatal, responsive to chemotherapy, but still
  incurable
• A member of a spectrum of plasma cell disorders
• A pre-malignant lesion called monoclonal
  gammopathy of undetermined significance (MGUS),
  which is present in 1 of adults, progresses to
  malignant MM at a rate of 1 per
• Karyotypes of MM are complex - Chr 13 loss
  common and poor prognostic factor
• Primary translocations involving 14q32 common
• 50of MGUS and MM
• Genetic changes are similar in MGUS and MM and
  amyloid
• All patients should be evaluated at academic
  center for enrollment in clinical trials

44
References

• 1. Avet-Loiseau H, Facon T, Grosbois B,
  Magrangeas F, Rapp MJ, Harousseau JL, Minvielle
  S, Bataille R. Oncogenesis of multiple myeloma
  14q32 and 13q chromosomal abnormalities are not
  randomly distributed, but correlate with natural
  history, immunological features, and clinical
  presentation. Blood. 2002992185-2191
• 2. Avet-Loiseau H, Garand R, Lode L, Harousseau
  JL, Bataille R. Translocation t(1114)(q13q32)
  is the hallmark of IgM, IgE, and nonsecretory
  multiple myeloma variants. Blood.
  20031011570-1571
• 3. Bergsagel PL, Chesi M, Nardini E, Brents LA,
  Kirby SL, Kuehl WM. Promiscuous translocations
  into immunoglobulin heavy chain switch regions in
  multiple myeloma. Proc Natl Acad Sci U S A.
  19969313931-13936
• 4. Bezieau S, Devilder MC, Avet-Loiseau H,
  Mellerin MP, Puthier D, Pennarun E, Rapp MJ,
  Harousseau JL, Moisan JP, Bataille R. High
  incidence of N and K-Ras activating mutations in
  multiple myeloma and primary plasma cell leukemia
  at diagnosis. Hum Mutat. 200118212-224
• 5. Chesi M, Nardini E, Brents LA, Schrock E,
  Ried T, Kuehl WM, Bergsagel PL. Frequent
  translocation t(414)(p16.3q32.3) in multiple
  myeloma is associated with increased expression
  and activating mutations of fibroblast growth
  factor receptor 3. Nat Genet. 199716260-264
• 6. Chesi M, Bergsagel PL, Shonukan OO, Martelli
  ML, Brents LA, Chen T, Schrock E, Ried T, Kuehl
  WM. Frequent dysregulation of the c-maf
  proto-oncogene at 16q23 by translocation to an Ig
  locus in multiple myeloma. Blood.
  1998914457-4463
• 7. Chesi M, Nardini E, Lim RS, Smith KD, Kuehl
  WM, Bergsagel PL. The t(414) translocation in
  myeloma dysregulates both FGFR3 and a novel gene,
  MMSET, resulting in IgH/MMSET hybrid transcripts.
  Blood. 1998923025-3034

45
References 2

• 8. Chesi M, Brents LA, Ely SA, Bais C, Robbiani
  DF, Mesri EA, Kuehl WM, Bergsagel PL. Activated
  fibroblast growth factor receptor 3 is an
  oncogene that contributes to tumor progression in
  multiple myeloma. Blood. 200197729-736
• 9. Davies FE, Dring AM, Li C, Rawstron AC,
  Shammas MA, O'Connor SM, Fenton JA, Hideshima T,
  Chauhan D, Tai IT, Robinson E, Auclair D, Rees K,
  Gonzalez D, Ashcroft AJ, Dasgupta R, Mitsiades C,
  Mitsiades N, Chen LB, Wong WH, Munshi NC, Morgan
  GJ, Anderson KC. Insights into the multistep
  transformation of MGUS to myeloma using
  microarray expression analysis. Blood. 2003
• 10. Facon T, Avet-Loiseau H, Guillerm G, Moreau
  P, Genevieve F, Zandecki M, Lai JL, Leleu X,
  Jouet JP, Bauters F, Harousseau JL, Bataille R,
  Mary JY. Chromosome 13 abnormalities identified
  by FISH analysis and serum beta2-microglobulin
  produce a powerful myeloma staging system for
  patients receiving high-dose therapy. Blood.
  2001971566-1571
• 11. Fonseca R, Witzig TE, Gertz MA, Kyle RA,
  Hoyer JD, Jalal SM, Greipp PR. Multiple myeloma
  and the translocation t(1114)(q13q32) a report
  on 13 cases. Br J Haematol. 1998101296-301
• 12. Fonseca R, Ahmann GJ, Jalal SM, Dewald GW,
  Larson DR, Therneau TM, Gertz MA, Kyle RA, Greipp
  PR. Chromosomal abnormalities in systemic
  amyloidosis. Br J Haematol. 1998103704-710
• 13. Fonseca R, Oken MM, Greipp PR. The
  t(414)(p16.3q32) is strongly associated with
  chromosome 13 abnormalities in both multiple
  myeloma and monoclonal gammopathy of undetermined
  significance. Blood. 2001981271-1272
• 14. Fonseca R, Debes-Marun CS, Picken EB, Dewald
  GW, Bryant SC, Winkler JM, Blood E, Oken MM,
  Santana-Davila R, Gonzalez-Paz N, Kyle RA, Gertz
  MA, Dispenzieri A, Lacy MQ, Greipp PR. The
  recurrent IgH translocations are highly
  associated with nonhyperdiploid variant multiple
  myeloma. Blood. 20031022562-2567

46
References 3

• 15. Fonseca R, Blood E, Rue M, Harrington D,
  Oken MM, Kyle RA, Dewald GW, Van Ness B, Van Wier
  SA, Henderson KJ, Bailey RJ, Greipp PR. Clinical
  and biologic implications of recurrent genomic
  aberrations in myeloma. Blood. 20031014569-4575
• 16. Hideshima T, Anderson KC. Molecular
  mechanisms of novel therapeutic approaches for
  multiple myeloma. Nat Rev Cancer. 20022927-937
• 17. Kuehl WM, Bergsagel PL. Multiple myeloma
  evolving genetic events and host interactions.
  Nat Rev Cancer. 20022175-187
• 18. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy
  MQ, Dispenzieri A, Fonseca R, Rajkumar SV, Offord
  JR, Larson DR, Plevak ME, Therneau TM, Greipp PR.
  Review of 1027 patients with newly diagnosed
  multiple myeloma. Mayo Clin Proc. 20037821-33
• 19. Li Z, Zhu YX, Plowright EE, Bergsagel PL,
  Chesi M, Patterson B, Hawley TS, Hawley RG,
  Stewart AK. The myeloma-associated oncogene
  fibroblast growth factor receptor 3 is
  transforming in hematopoietic cells. Blood.
  2001972413-2419
• 20. Magrangeas F, Nasser V, Avet-Loiseau H,
  Loriod B, Decaux O, Granjeaud S, Bertucci F,
  Birnbaum D, Nguyen C, Harousseau JL, Bataille R,
  Houlgatte R, Minvielle S. Gene expression
  profiling of multiple myeloma reveals molecular
  portraits in relation to the pathogenesis of the
  disease. Blood. 20031014998-5006
• 21. Malgeri U, Baldini L, Perfetti V, Fabris S,
  Vignarelli MC, Colombo G, Lotti V, Compasso S,
  Bogni S, Lombardi L, Maiolo AT, Neri A. Detection
  of t(414)(p16.3q32) chromosomal translocation
  in multiple myeloma by reverse transcription-polym
  erase chain reaction analysis of IGH-MMSET fusion
  transcripts. Cancer Res. 2000604058-4061

47
References 4

• 22. Moreau P, Facon T, Leleu X, Morineau N,
  Huyghe P, Harousseau JL, Bataille R, Avet-Loiseau
  H. Recurrent 14q32 translocations determine the
  prognosis of multiple myeloma, especially in
  patients receiving intensive chemotherapy. Blood.
  20021001579-1583
• 23. Onwuazor ON, Wen XY, Wang DY, Zhuang L,
  Masih-Khan E, Claudio J, Barlogie B, Shaughnessy
  JD, Jr., Stewart AK. Mutation, SNP, and isoform
  analysis of fibroblast growth factor receptor 3
  (FGFR3) in 150 newly diagnosed multiple myeloma
  patients. Blood. 2003102772-773
• 24. Perfetti V, Coluccia AM, Intini D, Malgeri
  U, Vignarelli MC, Casarini S, Merlini G, Neri A.
  Translocation T(414)(p16.3q32) is a recurrent
  genetic lesion in primary amyloidosis. Am J
  Pathol. 20011581599-1603
• 25. Plowright EE, Li Z, Bergsagel PL, Chesi M,
  Barber DL, Branch DR, Hawley RG, Stewart AK.
  Ectopic expression of fibroblast growth factor
  receptor 3 promotes myeloma cell proliferation
  and prevents apoptosis. Blood. 200095992-998
• 26. Santra M, Zhan F, Tian E, Barlogie B,
  Shaughnessy J, Jr. A subset of multiple myeloma
  harboring the t(414)(p16q32) translocation
  lacks FGFR3 expression but maintains an IGH/MMSET
  fusion transcript. Blood. 20031012374-2376
• 27. Sharathkumar A, Kirby M, Freedman M,
  Abdelhaleem M, Chitayat D, Teshima IE, Dror Y.
  Malignant hematological disorders in children
  with Wolf-Hirschhorn syndrome. Am J Med Genet.
  2003119A194-199
• 28. Shaughnessy J, Jr., Gabrea A, Qi Y, Brents
  L, Zhan F, Tian E, Sawyer J, Barlogie B,
  Bergsagel PL, Kuehl M. Cyclin D3 at 6p21 is
  dysregulated by recurrent chromosomal
  translocations to immunoglobulin loci in multiple
  myeloma. Blood. 200198217-223

48
References 5

• 29. Tarte K, Zhan F, De Vos J, Klein B,
  Shaughnessy J, Jr. Gene expression profiling of
  plasma cells and plasmablasts toward a better
  understanding of the late stages of B-cell
  differentiation. Blood. 2003102592-600
• 30. Winkler JM, Greipp P, Fonseca R.
  t(414)(p16.3q32) is strongly associated with a
  shorter survival in myeloma patients. Br J
  Haematol. 2003120170-171
• 31. Zhan F, Hardin J, Kordsmeier B, Bumm K,
  Zheng M, Tian E, Sanderson R, Yang Y, Wilson C,
  Zangari M, Anaissie E, Morris C, Muwalla F, van
  Rhee F, Fassas A, Crowley J, Tricot G, Barlogie
  B, Shaughnessy J, Jr. Global gene expression
  profiling of multiple myeloma, monoclonal
  gammopathy of undetermined significance, and
  normal bone marrow plasma cells. Blood.
  2002991745-1757
• 32. Zojer N, Konigsberg R, Ackermann J, Fritz E,
  Dallinger S, Kromer E, Kaufmann H, Riedl L,
  Gisslinger H, Schreiber S, Heinz R, Ludwig H,
  Huber H, Drach J. Deletion of 13q14 remains an
  independent adverse prognostic variable in
  multiple myeloma despite its frequent detection
  by interphase fluorescence in situ hybridization.
  Blood. 2000951925-1930