1. Illegitimate WNT signaling promotes 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

2. What is Multiple Myeloma? Multiple myeloma (also known as myeloma or plasma cell myeloma) is a progressive hematologic (blood) disease.Multiple myeloma (also known as myeloma or plasma cell myeloma) is a progressive hematologic (blood) disease. It is a cancer of the plasma cell, an important part of the immune system that produces immunoglobulins, or antibodies, to help fight infection and disease.It is a cancer of the plasma cell, an important part of the immune system that produces immunoglobulins, or antibodies, to help fight infection and disease. Multiple myeloma is characterized by excessive numbers of tumor cells that expand in the bone marrow leading to pancytopenia, an abnormal reduction in the number of RBC’s, WBC’s and blood platelets in the blood.Multiple myeloma is characterized by excessive numbers of tumor cells that expand in the bone marrow leading to pancytopenia, an abnormal reduction in the number of RBC’s, WBC’s and blood platelets in the blood. Overproduction of intact monoclonal immunoglobulin (IgG, IgA, IgD, or IgE) or Bence-Jones protein (free monoclonal k and L light chains) also occurs.Overproduction of intact monoclonal immunoglobulin (IgG, IgA, IgD, or IgE) or Bence-Jones protein (free monoclonal k and L light chains) also occurs. Transformation of a normal B cell into a malignant plasma cell involves a multi-step process that includes multiple genetic abnormalities.Transformation of a normal B cell into a malignant plasma cell involves a multi-step process that includes multiple genetic abnormalities.

3. Resulting plasma cells become malignant, meaning they continue to divide unchecked, generating more plasma cells that are malignant. These myeloma cells travel through the bloodstream and collect in the bone marrow, where they damage tissue. Hypercalcemia, anemia, renal damage, increased susceptibility to bacterial infection, and impaired production of normal immunoglobulin are common clinical manifestations of multiple myeloma. Most of the evolution takes place in the bone marrow indicating that signals from the bone marrow microenvironment, which may include paracrine growth factors, play a critical role in sustaining the growth and survival of MM cells during tumor progression.

4. It is often also characterized by diffuse osteoporosis, usually in the pelvis, spine, ribs, and skull. It is often also characterized by diffuse osteoporosis, usually in the pelvis, spine, ribs, and skull. Multiple myeloma is the second most prevalent blood cancer after non- Hodgkin's lymphoma. It represents approximately 1% of all cancers and 2% of all cancer deaths. Multiple myeloma is the second most prevalent blood cancer after non- Hodgkin's lymphoma. It represents approximately 1% of all cancers and 2% of all cancer deaths. The median age at diagnosis is about 71 years, and only 2% of cases are diagnosed in individuals under the age of 45. The median age at diagnosis is about 71 years, and only 2% of cases are diagnosed in individuals under the age of 45. Approximately 50,000 Americans currently have myeloma, and the American Cancer Society estimates that approximately 15,270 new cases of myeloma will be diagnosed during 2004. Approximately 50,000 Americans currently have myeloma, and the American Cancer Society estimates that approximately 15,270 new cases of myeloma will be diagnosed during 2004. 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 been identified. 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 been identified. Information found at www.multiplemyeloma.org

5. Authors Questions Do myeloma cells overexpress  -catenin and nonphosphorylated  - catenin? Do  -catenin and nonphosphorylated  -catenin get overexpressed in the primary myelomas in the bone marrow? Can Multiple Myeloma cells respond to WNT signaling? Does WNT signaling affect the localization of  -catenin? 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?

6. 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

7. 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.

8. 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.

9. Do myeloma cells overexpress  -catenin and non- phosphorylated  -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

10. Do  -catenin and non-phosphorylated  -catenin get 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

11. 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

12. Fig. 2b LiCl induces accumulation of  -catenin and increased nuclear localization of nonphosphorylated  -catenin. Apart from causing  -catenin accumulation, WNT signaling also affected the localization of  -catenin. Apart from causing  -catenin accumulation, WNT signaling also affected the localization of  -catenin. Before LiCl stimulation, low amounts of  -catenin were detected in the cytoplasm and nucleus of MM cells by confocal laser scan microscopy. Before LiCl stimulation, low amounts of  -catenin were detected in the cytoplasm and nucleus of MM cells by confocal laser scan microscopy. Non-phos  -catenin  -catenin Does WNT Signaling affect the localization of  -catenin?

13. Stimulation with LiCl led to an increase in the total amount of  -catenin (located to the plasma membrane at cell-cell contact sites,) as well as in the nucleus Stimulation with LiCl led to an increase in the total amount of  -catenin (located to the plasma membrane at cell-cell contact sites,) as well as in the nucleus Most of the nonphosphorylated  -catenin was localized in the nucleus. Most of the nonphosphorylated  -catenin was localized in the nucleus. Fig. 2b LiCl induces accumulation of  -catenin and increased nuclear localization of nonphosphorylated  -catenin. Yes, WNT signaling affects the localization of  -catenin

14. Figure 2c. Expression and nuclear localization of  -catenin in primary MM’s. Nonphosphorylated  -catenin was present in the nucleus of primary myelomas. Nonphosphorylated  -catenin was present in the nucleus of primary myelomas. Nuclear localization of nonphosphorylated  -catenin was also detected in primary myelomas, but the levels varied among individual cells within a given tumor. Nuclear localization of nonphosphorylated  -catenin was also detected in primary myelomas, but the levels varied among individual cells 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?

15. Fig 3a. Exogenous Wnt3a promotes proliferation of MM cells. MM cell lines Myeloma cells responded to stimulation with Wnt3a-conditioned medium with a 2- to 4-fold increase in proliferation and was readily observed within the first 24 hours. Myeloma cells responded to stimulation with Wnt3a-conditioned medium with a 2- to 4-fold increase in proliferation and was readily observed within the first 24 hours. Cell lines were cultured in the presence of L cell- conditioned medium (white bars) or conditioned medium derived from Wnt3a-transfected L cells (black bars) Cell lines were cultured in the presence of L cell- conditioned medium (white bars) or conditioned medium derived from Wnt3a-transfected L cells (black bars) [ 3 H] thymidine incorporation was measured after 1, 2, and 3 days of culture. [ 3 H] thymidine incorporation was measured after 1, 2, and 3 days of culture. Does WNT signaling control the proliferation of myeloma cells?

16. Figure 3b. Purified Wnt3a promotes proliferation of MM cells Cells were cultured in the absence or presence of purified Wnt3a in serum-free medium. Cells were cultured in the absence or presence of purified Wnt3a in serum-free medium. [ 3 H] thymidine incorporation was measured after 2 days of culture. [ 3 H] thymidine incorporation was measured after 2 days of culture. Similar results were also obtained with purified Wnt3a, demonstrating 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 Wnt3a-transfected L cells. Similar results were also obtained with purified Wnt3a, demonstrating 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 Wnt3a-transfected L cells. MM cell lines Does WNT signaling control the proliferation of myeloma cells?

17. Fig. 3c. LiCl stimulation promotes proliferation of MM cells. Cells were cultured in the absence or presence of 2mM LiCl in serum-free medium. Cells were cultured in the absence or presence of 2mM LiCl in serum-free medium. [ 3 H] thymidine incorporation was measured after 3 days of culture. [ 3 H] thymidine incorporation was measured after 3 days of culture. Treatment with LiCl (which inhibited GSK3  resulting in  -catenin accumulation Fig 2a.) gave rise to a similar increase in proliferation. Treatment with LiCl (which inhibited GSK3  resulting in  -catenin accumulation Fig 2a.) gave rise to a similar increase in proliferation. MM cell lines Does WNT signaling control the proliferation of myeloma cells?

18. Fig. 3d  -catenin S33Y promotes proliferation of MM cells. Cells were transfected with empty vector or with  -catenin S33Y Cells were transfected with empty vector or with  -catenin S33Y [ 3 H] thymidine incorporation was measured after 2 days of culture. [ 3 H] thymidine incorporation was measured after 2 days of culture. Enhanced proliferation was also observed after expression of the  - catenin S33Y mutant. Enhanced proliferation was also observed after expression of the  - catenin S33Y mutant. MM cell lines Yes, WNT signaling controls the proliferation of Myeloma cells Does WNT signaling control the proliferation of myeloma cells?

19. Fig. 4a  TCF4 inhibits proliferation of MM cells MM cell lines OPM1 and NCI H929 were transfected with either  TCF or empty vector, both in combo with pEGFP constructs MM cell lines OPM1 and NCI H929 were transfected with either  TCF or empty vector, both in combo with pEGFP constructs After overnight culture, viable GFP-positive cells were sorted and [ 3 H] thymidine incorporation was measured after 2 and 3 days of culture. After overnight culture, viable GFP-positive cells were sorted and [ 3 H] thymidine incorporation was measured after 2 and 3 days of culture. Transfection with  TCF4 (a dominant negative form of TCF) strongly inhibited proliferation of the MM cell lines OPM1 and NCI H929, which both contain large amounts of active  -catenin. Transfection with  TCF4 (a dominant negative form of TCF) strongly inhibited proliferation of the MM cell lines OPM1 and NCI H929, which both contain large amounts of active  -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

20. This suggests…. WNT signaling is constitutively active, but not maximally activated and sensitive to regulation. So to corroborate these conclusions…..

21. Fig. 4b TCF reporter activity in MM cells. OPM1 cells were either transfected with luciferase reporter constructs (pTOPFLASH), alone or in combo with  -catenin S33Y and  TCF4 and were assayed for luciferase activity. OPM1 cells were either transfected with luciferase reporter constructs (pTOPFLASH), alone or in combo with  -catenin S33Y and  TCF4 and were assayed for luciferase activity. OPM1 showed a moderate constitutive  -catenin/TCF activity. OPM1 showed a moderate constitutive  -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.

22. Fig. 4c Wnt3a stimulates TCF reported activity in MM cells. OPM1 and NCI H929 cells were transfected with pTOPFLASH, alone or in combo with b-catenin S33Y. OPM1 and NCI H929 cells were transfected with pTOPFLASH, alone or in combo with b-catenin S33Y. The cells were either stimulated or not stimulated with purified Wnt3a, and were assayed for luciferase activity. The cells were either stimulated or not stimulated with purified Wnt3a, and were assayed for luciferase activity. TCF reporter activity was increased by stimulating MM cells with purified Wnt3a TCF reporter activity was increased by stimulating MM cells with purified Wnt3a Yes, the WNT pathway in MM cells are constitutively active

23. Is there a regulatory component? (the presence of an autocrine activation loop?)

24. Table 1. WNT Expression in myelomas and normal B lineage cells Neither normal B cells nor plasma cells expressed these WNT’s Neither 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.

25. Table 1. Continued…. Expression of WNT5a and WNt10b were found in all myeloma cell lines tested, but only WNT16 transcripts were found in one MM cell line. Expression of WNT5a and WNt10b were found in all myeloma cell lines tested, but only WNT16 transcripts were found in one MM cell line.

26. Table 1. Continued…. In highly purified MM cells, they detected expression of WNT5a and/or WNT10b and, in one case, WNT16 In highly purified MM cells, they detected expression of WNT5a and/or WNT10b and, in one case, WNT16

27. Table 1. Continued…. WNT5a and WNT10b expression was also found in bone marrow stromal cells suggesting they may function as a paracrine source of WNT’s within the bone marrow microenvironment WNT5a and WNT10b expression was also found in bone marrow stromal cells suggesting they may function as a paracrine source of WNT’s within the bone marrow microenvironment

28. Finding 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)

29. 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

30. 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