Illegitimate WNT signaling promotes Illegitimate WNT signaling promotes proliferation of multiple myeloma cells.proliferation of multiple myeloma cells.

Patrick W. B. Derksen*, Esther Tjin*, Helen P. Meijer*, Melanie D. Klok*, Harold D. Mac Gillavry*, Marinus H. J. van Oers†, Henk M. Lokhorst‡, Andries C. Bloem§, Hans Clevers¶, Roel Nusse, Ronald van der Neut*, Marcel Spaargaren*, and Steven T. Pals*,**

Proceedings of the National Academy of Science (PNAS)April 20 , 2004

What is Multiple Myeloma?What is Multiple Myeloma?

• Multiple myeloma (also known as myeloma or plasma cell myeloma) is Multiple myeloma (also known as myeloma or plasma cell myeloma) is a progressive hematologic (blood) disease. a progressive hematologic (blood) disease.

• It is a cancer of the plasma cell, an important part of the immune It is a cancer of the plasma cell, an important part of the immune system that produces immunoglobulins, or antibodies, to help fight system that produces immunoglobulins, or antibodies, to help fight infection and disease.infection and disease.

• Multiple myeloma is characterized by excessive numbers of tumor Multiple myeloma is characterized by excessive numbers of tumor cells that expand in the bone marrow leading to pancytopenia, an cells that expand in the bone marrow leading to pancytopenia, an abnormal reduction in the number of RBC’s, WBC’s and blood abnormal reduction in the number of RBC’s, WBC’s and blood platelets in the blood. platelets in the blood.

• Overproduction of intact monoclonal immunoglobulin (IgG, IgA, IgD, or Overproduction of intact monoclonal immunoglobulin (IgG, IgA, IgD, or IgE) or Bence-Jones protein (free monoclonal k and L light chains) IgE) or Bence-Jones protein (free monoclonal k and L light chains) also occurs. also occurs.

• Transformation of a normal B cell into a malignant plasma cell involves Transformation of a normal B cell into a malignant plasma cell involves a multi-step process that includes multiple genetic abnormalities.a multi-step process that includes multiple genetic abnormalities.

Resulting plasma cells become malignant, meaning they continue to divide Resulting plasma cells become malignant, meaning they continue to divide unchecked, generating more plasma cells that are malignant.unchecked, generating more plasma cells that are malignant.

These myeloma cells travel through the bloodstream and collect in the bone These myeloma cells travel through the bloodstream and collect in the bone marrow, where they damage tissue.marrow, where they damage tissue.

Hypercalcemia, anemia, renal damage, increased susceptibility to bacterial Hypercalcemia, anemia, renal damage, increased susceptibility to bacterial infection, and impaired production of normal immunoglobulin are common infection, and impaired production of normal immunoglobulin are common clinical manifestations of multiple myeloma.clinical manifestations of multiple myeloma.

Most of the evolution takes place in the bone Most of the evolution takes place in the bone marrow indicating that signals from the bone marrow indicating that signals from the bone marrow microenvironment, which may include marrow microenvironment, which may include paracrine growth factors, play a critical role in paracrine growth factors, play a critical role in sustaining the growth and survival of MM cells sustaining the growth and survival of MM cells during tumor progression.during tumor progression.

• It is often also characterized by diffuse osteoporosis, usually in the It is often also characterized by diffuse osteoporosis, usually in the pelvis, spine, ribs, and skull. pelvis, spine, ribs, and skull.

• Multiple myeloma is the second most prevalent blood cancer after non-Multiple myeloma is the second most prevalent blood cancer after non-Hodgkin's lymphoma. It represents approximately 1% of all cancers Hodgkin's lymphoma. It represents approximately 1% of all cancers and 2% of all cancer deaths. and 2% of all cancer deaths.

• The median age at diagnosis is about 71 years, and only 2% of cases The median age at diagnosis is about 71 years, and only 2% of cases are diagnosed in individuals under the age of 45. are diagnosed in individuals under the age of 45.

• Approximately 50,000 Americans currently have myeloma, and the Approximately 50,000 Americans currently have myeloma, and the American Cancer Society estimates that approximately 15,270 new American Cancer Society estimates that approximately 15,270 new cases of myeloma will be diagnosed during 2004. cases of myeloma will be diagnosed during 2004.

• Although a tremendous amount of work has gone into the search for Although a tremendous amount of work has gone into the search for the cause of multiple myeloma, to date no cause for this disease has the cause of multiple myeloma, to date no cause for this disease has been identified.been identified.

Information found at www.multiplemyeloma.org

Authors QuestionsAuthors Questions

Do myeloma cells overexpress Do myeloma cells overexpress -catenin and nonphosphorylated -catenin and nonphosphorylated --catenin?catenin?

Do Do -catenin and nonphosphorylated -catenin and nonphosphorylated -catenin get overexpressed in the -catenin get overexpressed in the primary myelomas in the bone marrow?primary myelomas in the bone marrow?

Can Multiple Myeloma cells respond to WNT signaling?Can Multiple Myeloma cells respond to WNT signaling?

Does WNT signaling affect the localization of Does WNT signaling affect the localization of -catenin?-catenin?

Does WNT signaling control the proliferation of myeloma cells?Does WNT signaling control the proliferation of myeloma cells?

Does the disruption of -catenin/TCF activity effect Multiple Myeloma proliferation?

Is the WNT pathway in MM cells intact?

Is there a regulatory component?

• WNT signals are one class of paracrine growth factors that could act to influence the growth of MM cells

• WNT signal transduction components, in particular adenomatous polyposis coli (APC), and -catenin, are often mutated in cancers and sustained overexpression of WNT genes can cause cancer.

• WNT proteins themselves are able to promote the proliferation of progenitor or stem cells

• The main event in the WNT signaling pathway is the stabilization of -catenin

• Signaling by WNT factors blocks GSK3 activity, resulting in the accumulation of nonphosphorylated -catenin, which will translocate to the nucleus

WNT signaling

• Once in the nucleus non phosphorylated -catenin joins T cell factor (TCF) transcription factors to drive transcription of target genes

• It has been shown that uncontrolled -catenin/ TCF activity plays a big role in many human cancers

• The involvement of the WNT pathway in the regulation of the survival and expansion of progenitor and stem cells, in combination with its oncogenic potential in nonlymphoid cells, made the authors want to test whether

deregulation of the WNT pathway occurs in lymphoid neoplasia.

Overview of WNT pathway

•Without WNT signaling, -catenin exists in its phosphorylated form which is tagged with ubiquitin and destroyed.

•With WNT signaling unphosphorylated-catenin is able to enter the nucleus, bind to TCF, displace groucho and drive transcription of several genes including c-myc.

Do myeloma cells overexpress Do myeloma cells overexpress -catenin and non--catenin and non-phosphorylated phosphorylated -catenin? -catenin?

•Figure 1A shows that all myeloma cell lines tested contained significant -catenin levels and most of them contained detectable levels of non-phosphorylated -catenin.

•Figure 1B compares normal B-cells and plasma cells to MM cells.

•This assay shows that there is a big difference between the expression of -catenin and nonphosphorylated -catenin in MM cells vs. normal B-cells and plasma cells.

Yes, myeloma cells do overexpress both forms of -catenin

Do Do -catenin and non-phosphorylated -catenin and non-phosphorylated -catenin get -catenin get overexpressed in primary myelomas in the bone marrow?overexpressed in primary myelomas in the bone marrow?

•Figure 1C demonstrates that -catenin is expressed in 9 out of 10 primary MM bone marrow samples

•Non-phosphorylated -catenin is also detectable in most primary MM bone marrow samples

•Figure 1D was used to show that 7 out of 7 normal bone marrow samples do not express -catenin or non-phosphorylated -catenin

Yes, both forms of -catenin are overexpressed in primary myelomas

Can MM cells respond to WNT signaling?Can MM cells respond to WNT signaling?

•Stimulation with LiCl resulted in increased -catenin and non-phosphorylated -catenin

•Stimulation with Wnt3a (a specific Wnt protein) produced similar results

Yes, myeloma cells do respond to Wnt signaling

Fig. 2b LiCl induces accumulation of Fig. 2b LiCl induces accumulation of -catenin and increased nuclear localization -catenin and increased nuclear localization of nonphosphorylated of nonphosphorylated -catenin.-catenin.

• Apart from causing Apart from causing -catenin -catenin accumulation, WNT accumulation, WNT signaling also affected the signaling also affected the localization of localization of -catenin. -catenin.

• Before LiCl stimulation, low Before LiCl stimulation, low amounts of amounts of -catenin were -catenin were detected in the cytoplasm detected in the cytoplasm and nucleus of MM cells by and nucleus of MM cells by confocal laser scan confocal laser scan microscopy. microscopy.

Non-phos -catenin

-catenin

Does WNT Signaling affect the localization of -catenin?

Does WNT Signaling affect the localization of Does WNT Signaling affect the localization of -catenin?-catenin?

• Stimulation with LiCl led to an Stimulation with LiCl led to an increase in the total amount of increase in the total amount of -catenin (located to the -catenin (located to the plasma membrane at cell-cell plasma membrane at cell-cell contact sites,) as well as in the contact sites,) as well as in the nucleusnucleus

• Most of the nonphosphorylated Most of the nonphosphorylated -catenin was localized in the -catenin was localized in the nucleus.nucleus.

Fig. 2b LiCl induces accumulation of -catenin and increased nuclear localization of nonphosphorylated -catenin.

Yes, WNT signaling affects the localization of -catenin

Figure 2c. Expression and nuclear localization of Figure 2c. Expression and nuclear localization of -catenin in primary MM’s.-catenin in primary MM’s. • Nonphosphorylated Nonphosphorylated -catenin was present in the nucleus of primary -catenin was present in the nucleus of primary

myelomas. myelomas. • Nuclear localization of nonphosphorylated Nuclear localization of nonphosphorylated -catenin was also detected -catenin was also detected

in primary myelomas, but the levels variedin primary myelomas, but the levels varied among individual cells among individual cells within a given tumor.within a given tumor.

Nuclear staining of non-phos -catenin(blue)

Isotype-matched antibody in combo with the anti-Ig light-chain antibodies

Control

Yes, WNT signaling affects the localization of -catenin

Does WNT Signaling affect the localization of -catenin?

Fig 3a. Exogenous Wnt3a promotes proliferation of MM cells.Fig 3a. Exogenous Wnt3a promotes proliferation of MM cells. MM cell lines

• Myeloma cells responded to stimulation with Wnt3a-conditioned Myeloma cells responded to stimulation with Wnt3a-conditioned medium with a 2- to 4-fold increase in proliferation and was readily medium with a 2- to 4-fold increase in proliferation and was readily observed within the first 24 hours.observed within the first 24 hours.

• Cell lines were Cell lines were cultured in the cultured in the presence of L cell-presence of L cell-conditioned medium conditioned medium (white bars) or (white bars) or conditioned medium conditioned medium derived from Wnt3a-derived from Wnt3a-transfected L cells transfected L cells (black bars)(black bars)

• [[33H] thymidine H] thymidine incorporation was incorporation was measured after 1, 2, measured after 1, 2, and 3 days of culture.and 3 days of culture.

Does WNT signaling control the proliferation of myeloma cells?

Figure 3b. Purified Wnt3a promotes proliferation of MM cellsFigure 3b. Purified Wnt3a promotes proliferation of MM cells

• Cells were cultured in the Cells were cultured in the absence or presence of purified absence or presence of purified Wnt3a in serum-free medium. Wnt3a in serum-free medium.

• [[33H] thymidine incorporation was H] thymidine incorporation was measured after 2 days of measured after 2 days of culture.culture.

• Similar results were also obtained with purified Wnt3a, demonstrating Similar results were also obtained with purified Wnt3a, demonstrating the specificity of the effect of Wnt3a and ruling out indirect effects of the specificity of the effect of Wnt3a and ruling out indirect effects of other growth factors released as a result of autocrine stimulation in the other growth factors released as a result of autocrine stimulation in the Wnt3a-transfected L cells.Wnt3a-transfected L cells.

MM cell lines

Does WNT signaling control the proliferation of myeloma cells?

Fig. 3c.Fig. 3c. LiCl stimulation promotes proliferation of MM cells.LiCl stimulation promotes proliferation of MM cells.

• Cells were cultured in the Cells were cultured in the absence or presence of absence or presence of 2mM LiCl in serum-free 2mM LiCl in serum-free medium. medium.

• [[33H] thymidine incorporation H] thymidine incorporation was measured after 3 days was measured after 3 days of culture.of culture.

• Treatment with LiCl (which inhibited GSK3Treatment with LiCl (which inhibited GSK3resulting in resulting in -catenin -catenin accumulation Fig 2a.) gave rise to a similar increase in proliferation.accumulation Fig 2a.) gave rise to a similar increase in proliferation.

MM cell lines

Does WNT signaling control the proliferation of myeloma cells?

Fig. 3d Fig. 3d -catenin S33Y promotes proliferation of MM cells.-catenin S33Y promotes proliferation of MM cells.

• Cells were transfected Cells were transfected with empty vector or with empty vector or with with -catenin S33Y-catenin S33Y

• [[33H] thymidine H] thymidine incorporation was incorporation was measured after 2 days measured after 2 days of culture.of culture.

• Enhanced proliferation was also observed after expression of the Enhanced proliferation was also observed after expression of the --

catenin S33Y mutant.catenin S33Y mutant.

MM cell lines

Yes, WNT signaling controls the proliferation of Myeloma cells

Does WNT signaling control the proliferation of myeloma cells?

Fig. 4a Fig. 4a TCF4 inhibits proliferation of MM cellsTCF4 inhibits proliferation of MM cells

• MM cell lines OPM1 and MM cell lines OPM1 and NCI H929 were transfected NCI H929 were transfected with either with either TCF or empty TCF or empty vector, both in combo with vector, both in combo with pEGFP constructspEGFP constructs

• After overnight culture, After overnight culture, viable GFP-positive cells viable GFP-positive cells were sorted and [were sorted and [33H] H] thymidine incorporation was thymidine incorporation was measured after 2 and 3 measured after 2 and 3 days of culture.days of culture.

• Transfection with Transfection with TCF4 (a dominant negative form of TCF) strongly inhibited TCF4 (a dominant negative form of TCF) strongly inhibited proliferation of the MM cell lines OPM1 and NCI H929, which both contain large proliferation of the MM cell lines OPM1 and NCI H929, which both contain large

amounts of active amounts of active -catenin.-catenin.

MM cell lines

Does the disruption of -catenin/TCF activity effect Multiple Myeloma proliferation?

Yes, disruption of -catenin/TCF activity causes inhibition of MM proliferation

This suggests….This suggests….

WNT signaling is constitutively active, but not WNT signaling is constitutively active, but not maximally activated and sensitive to regulation. maximally activated and sensitive to regulation.

So to corroborate these conclusions…..So to corroborate these conclusions…..

Fig. 4b TCF reporter activity in MM cells.Fig. 4b TCF reporter activity in MM cells.

• OPM1 cells were either transfected OPM1 cells were either transfected with luciferase reporter constructs with luciferase reporter constructs (pTOPFLASH), alone or in combo with (pTOPFLASH), alone or in combo with -catenin S33Y and -catenin S33Y and TCF4 and were TCF4 and were assayed for luciferase activity.assayed for luciferase activity.

• OPM1 showed a moderate constitutive OPM1 showed a moderate constitutive -catenin/TCF activity. -catenin/TCF activity.

• This activity was inhibited by contransfection of TCF4

• A strong receptor activity was obtained after cotransfection of the active -catenin mutant S33Y.

Fig. 4c Wnt3a stimulates TCF reported activity in MM cells.Fig. 4c Wnt3a stimulates TCF reported activity in MM cells.

• OPM1 and NCI H929 cells OPM1 and NCI H929 cells were transfected with were transfected with pTOPFLASH, alone or in pTOPFLASH, alone or in combo with b-catenin combo with b-catenin S33Y.S33Y.

• The cells were either The cells were either stimulated or not stimulated or not stimulated with purified stimulated with purified Wnt3a, and were assayed Wnt3a, and were assayed for luciferase activity.for luciferase activity.

• TCF reporter activity was increased by stimulating MM cells with TCF reporter activity was increased by stimulating MM cells with purified Wnt3apurified Wnt3a

Yes, the WNT pathway in MM cells are constitutively active

Is there a regulatory component?Is there a regulatory component?

(the presence of an autocrine activation loop?)(the presence of an autocrine activation loop?)

Table 1. WNT Expression in myelomas and normal B lineage Table 1. WNT Expression in myelomas and normal B lineage cells cells

• Neither normal B cells nor plasma cells expressed these WNT’sNeither normal B cells nor plasma cells expressed these WNT’s

• To explore this possibility, the authors assessed the expression of WNT genes previously demonstrated to be expressed within the hematopoietic environment.

Table 1. Continued….Table 1. Continued….

• Expression of Expression of WNT5aWNT5a and and WNt10bWNt10b were found in all myeloma cell were found in all myeloma cell lines tested, but only lines tested, but only WNT16WNT16 transcripts were found in one MM cell transcripts were found in one MM cell line.line.

Table 1. Continued….Table 1. Continued….

• In highly purified MM cells, they detected expression of In highly purified MM cells, they detected expression of WNT5aWNT5a and/or and/or WNT10bWNT10b and, in one case, and, in one case, WNT16WNT16

Table 1. Continued….Table 1. Continued….

• WNT5aWNT5a and and WNT10bWNT10b expression was also found in bone marrow expression was also found in bone marrow stromal cells suggesting they may function as a paracrine source stromal cells suggesting they may function as a paracrine source of WNT’s within the bone marrow microenvironmentof WNT’s within the bone marrow microenvironment

Finding AnswersFinding Answers

1. Do myeloma cells overexpress -catenin and non phosphorylated -catenin?

Yes (see Fig 1A & B)

2. Do -catenin and nonphosphorylated -catenin get overexpressed in primary myelomas in the bone marrow?

Yes (see Fig 1C & D)

3. Can MM cells respond to Wnt Signaling?

Yes (see Fig 2A)4. Does Wnt Signaling affect the localization of -catenin?

Yes (see Fig 2B & C)

5. Does Wnt signaling control the proliferation of myeloma cells?

Yes (see Fig 3A-D)

Finding Answers Cont…Finding Answers Cont…

6. Does the disruption of -catenin/TCF activity effect Multiple Myeloma proliferation?

Yes (Figure 4a.)

7. Is the WNT pathway in MM cells intact?

Yes (Figures 4b & 4c.)

8. Is there a regulatory component?

Yes, WNt5a and WNt10b may function as a paracrine source of WNT’s within the bone marrow

microenvironment

Conclusions

•Since normal plasma cells are fully differentiated, the activation of the signaling routes that causes cell proliferation is important for their transformation into MM cells. -this study showed WNT signaling can control the proliferation of MM

•This means the WNT pathway may prove to be a good target for MM therapy.

•More research is needed to determine which WNT target genes might be involved in WNT induced proliferation in MM