Haematologica 2003 - Supplements

8. Mouse models for MM
214
Suppressor of Cytokine Signalling-3 (SOCS3) is a key
physiological negative regulator of IL-6
Andrew Roberts, Ben Croker, Danielle Krebs, Jian-Guo
Zhang, Sam Wormald, Don Metcalf, Nicos Nicola, Douglas
Hilton, Warren Alexander
The Walter and Eliza Hall Institute of Medical Research,
Melbourne, Australia.
Because IL-6 is a key modulator of myeloma cell survival and
proliferation, identification of intracellular negative regulators of
IL-6 signalling is an important step in the process of developing
novel therapies. The SOCS family of proteins are attractive
candidates as physiological regulators of IL-6 signalling. SOCS1
and SOCS3 are highly homologous and the expression of both is
induced by IL-6 in both lymphoid and myeloid cells. In
overexpression systems, both can inhibit STAT activation after
gp130 ligation by IL-6. However, IL-6 signalling is not perturbed
in primary cells from SOCS1 knockout mice. We have therefore
concentrated on investigating the role of SOCS3 in IL-6
signalling in primary haemopoietic cells.
A conditional gene targeting strategy was pursued because
SOCS3 knockout mice die early in utero of placental failure. We
have generated mice in which either haemopoietic or hepatic cells
have no functional SOCS3 alleles, by using mice bearing a Lox-P
flanked allele of SOCS3 (ki) and a null allele (o), and
intercrossing them with mice expressing Cre recombinase
selectively in haemopoietic cells (LysMCre or VavCre) or hepatic
cells (AlbCre) respectively.
Hepatocytes from AlbCre+SOCS3fl/o do not express SOCS3.
Similarly, macrophages from LysMCre+SOCS3fl/o mice and all
haemopoietic cells from VavCre+SOCS3fl/o mice are totally
deficient in SOCS3. In all SOCS3 deficient cells from these mice,
marked abnormalities in IL-6 signalling and cellular responses
were observed. Following either in vitro or in vivo stimulation
with IL-6, STAT3 phosphorylation was both increased and
prolonged. Microarray analysis confirmed this excess signalling
resulted in aberrant target gene transcription. Accordingly,
cellular responses to IL-6 were amplified in the absence of
SOCS3: (i) serum acute phase proteins were increased in
AlbCre+SOCS3fl/o mice; (ii) IL-6 inhibition of macrophage
proliferation was augmented; and (iii) myeloid progenitor cell
proliferation was increased.
These data unequivocally prove that SOCS3 is a key
physiological negative regulator of IL-6 signalling. Whether the
absence of SOCS3 increases plasmacytoma development or
progression in murine models is being addressed.
215
NOVEL TARGETED DEREGULATION OF APOPTOTIC
AND ONCOGENIC PATHWAYS LEADS TO
ACCELERATED B-LYMPHOID MALIGNANCY IN
GENETICALLY ENGINEERED MICE
Michael Linden* Wan-Cheung Cheung#, Sung Sup Park$,
Nicole Kirchhof*, Cathy Carlson* Kathy Pape#, Marc
Jenkins#, Roberto Polakiewicz#, Siegfried Janz$, and Brian
Van Ness*
#Cell Signaling Technologies (Beverly, MA), $National Cancer
Institute (Bethesda, MD), *University of Minnesota-Twin Cities
(Minneapolis, MN)
Multiple myeloma (MM) is characterized by the proliferation of
malignant plasma cells (PC’s) in the bone marrow. We have
undertaken a collaborative project to target gene deregulation that
will contribute to models of plasma cell malignancy in the mouse.
Specifically, our project seeks to address three goals: 1) Identify
novel transcriptional regulators whose activity is limited to B-
lymphoid cells in late developmental stages; 2) Develop new
mouse models of B-lymphoid malignancies, including MM, using
transcriptionally targeted transgenes; and 3) Develop novel
strategies to traffic plasmacytomagenesis in vivo. While no
pathognomonic lesion has been identified in MM, upregulation of
anti-apoptotic proteins (BCL-XL) and deregulation of growthpromoting
oncogenes (MYC and N-RAS) are common in many
MM patients. The 3' kappa immunoglobulin (Ig) light chain
enhancer (3’KE) regulates transcription of the kappa Ig gene, and
its activity in murine B-cells is restricted to the late stages of B-
cell development. The 3’KE was used to create a 3’KE/BCL-XL
transgenic mouse. It was expected that this transgene would alter
B-cell compartment composition, as the B-cells in late
developmental stages would be rendered resistant to apoptosis.
Indeed, we found significant increases in these cell populations,
and nests of PC’s were found in the bone marrow of 3’KE/BCL-
XL mice. Additionally, perivascular foci of PC’s with nuclear
atypia occur in multiple soft tissues in aged 3’KE/BCL-XL mice,
and other sequelae consistent with excess Ig production are
common. To accelerate B-lymphoid malignancies, we crossed the
3’KE/BCL-XL mouse to an E/MYC or E/MYC transgenic
mouse (these mice use the IgH E or E enhancers to drive
MYC expression). While Eµ activity begins early in B-cell
development and continues throughout all other developmental
stages, Eα activity is developmentally restricted to late stages of
development and is especially influential in governing heavy
chain Ig expression in PC’s. Co-expression of BCL-XL and
MYC under the context of the 3’KE and the E leads to a highly
fatal B-lymphoid malignancy with a median survival of 5.5
weeks. When MYC expression is controlled by the E,
however, fatal PC neoplasms in the bone marrow and other
lymphoid organs develop with a median survival of 14.5 weeks.
To facilitate visualization of normal and malignant PC’s, we are
employing the use of the PC-GFP mouse, which uses kappa Ig
regulatory elements to drive GFP expression in PC’s. To
investigate the role of constitutively activated RAS expression in
B-lymphoid malignancy, we have generated a 3’KE/N-RASV12
mouse. To elicit antigen-induced clonal expansion of the
genetically altered plasma cells, we are immunizing the
genetically engineered mice. In summary, deregulating apoptotic
and oncogenic pathways in plasma and other B-cells by using
novel transcriptional regulators in genetically engineered mice
serves as a good platform to further the understanding of B-
lymphoid malignancies, including MM.
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