Pathology

Siegried Janz, MD
Research Laboratory

Project 1 – Genetics of plasma-cell tumors

Unprecedented progress in our understanding of the biologic and molecular genetic underpinnings of human plasma-cell neoplasms (PCN) has led to the development of novel targeted therapeutic agents – including proteasome blockers (bortezomib), immunomodulatory agents (lenalidomide), therapeutic antibodies, and a variety of emerging small-drug inhibitors of cellular signal transduction pathways – that are now beginning to produce tangible benefits for patients with PCN. Nonetheless, the prognosis and outcome of PCN remain grim, caused in large measure by serious limitations in our knowledge of the genetic pathways of malignant plasma-cell (PC) transformation.

The research project, entitled, "Defining genetic pathways of plasma-cell neoplasia," seeks to address outstanding questions on the genetics of PCN in order to facilitate ongoing efforts by the clinical research community and the pharmacological industry to treat and prevent these malignancies more effectively. Using two powerful, complementary, unbiased approaches to genetic forward screening in a newly developed mouse model of human PCN, designated iMyc^ΔΕμ, we will first identify candidate driver genes involved in PC tumor progression in mice, and then evaluate these genes across the mouse-human species barrier to identify and validate the involvement of orthologous human driver genes in human PCN. To accomplish this goal, we propose three innovative, closely interrelated, specific research aims.

Aim 1 is to identify PCN driver genes in a virus-based forward genetic screen in mice. The rationale for this aim is based in part on findings from cancer screens, showing that the Moloney-based murine leukemia virus, MOL4070LTR, accelerates PC tumors in strain C.iMyc^ΔΕμ mice – a gene-insertion model of the Myc-activating T(12;15)(Igh-Myc) translocation. Aim 2 is to identify PCN driver genes in a transposon-based forward genetic screen in mice. The rationale for this aim develops from evidence that the Sleeping Beauty (SB) transposon-based cancer screen provides a flexible, efficient gene discovery tool that complements virus-dependent screens; e.g., by its applicability to quiescent cells such as PCs. Aim 3 is to validate the involvement of human orthologs of the putative driver genes identified in Aims 1 and 2 in human PCN, and the potential of these genes as new targets for cancer therapy and prevention (Fig. 1).

Figure 1: Premalignant conditions in MM, WM and CLL (panel A) and experimental strategy to elucidate genetic tumor progression pathways in Myc-transgenic mice of great relevance for human MM/WM (panel B).
(A) MGUS (IgG- or IgA-producing in the vast majority of cases), IgM MGUS and MBL represent the earliest detectable stages in the manifestation of MM, WM and B-CLL (chronic lymphocytic leukemia), respectively. The three premalignant conditions can be viewed as suspended states in the continuum from premalignant alteration to malignant disease. Alternatively, it has recently been proposed that MGUS should be viewed as a malignant tumor that is kept at bay by an ongoing immune response.
(B) We use iMyc-transgenic mice, which are prone to malignant B-cell tumors sharing important features with human MM and WM, in two complementary forward-genetic screens to unravel the genetic pathways of tumor progression.

Project 2 – Mouse model of Waldenström's macroglobulinemia

Transgenic mouse models of human cancer are experimental model systems that rely on laboratory mice that have been genetically manipulated to render them prone to neoplasms that accurately recapitulate important features of their human cancer counterparts. Model systems of this sort: enable researchers to study the onset and progression of cancer in ways that cannot be pursued in human beings; advance our understanding of the molecular genetic and biological events that contribute to the development and spread of cancer cells; and provide a valuable preclinical platform for evaluating new approaches to treat and prevent cancer in patients. The latter is particularly important in circumstances in which drug testing requires an intact, immunocompetent animal that is able to produce the same kind of tumor microenvironment and recruit the same types of tumor bystander cells commonly found in human patients. To give but one example, therapeutic antibodies target cancer cells by recruiting normal immune cells to the site of attack; thus, the preclinical testing of these antibodies requires strains of laboratory mice that have a normal, fully functioning immune system. The development of an immunocompetent, transgenic mouse model of human WM that will be useful for preclinical testing of WM drug candidates is the main objective of this research. To that end, we are generating a designer model of human WM designated C.IL6/BCL2/AID^null. This model combines three crucial pathogenetic factors of human WM – namely the B-lymphocyte growth, differentiation and survival factor IL-6, the cellular oncoprotein BCL-2, and the inability of WM cells to perform immunoglobulin isotype switching (AID^null) – on the genetic backgroud of BALB/c (abbreviated as C). Strain C mice are highly susceptible to malignant B-lymphocyte transformation (Fig. 2). 

Figure 2: Schematic overview of the pathogenesis of lymphoplasmacytic lymphoma (LPL)-WM (panel A) and transgenic mouse strains that we propose to use for modeling human LPL-WM in mice (panel B)
(A) IL6 and BCL2 have been identified as "WM genes" – genes that confer genetic proclivity to the disease (left). Additionally, IL-6 and BCL-2 are major player in the LPL-WM cells (right)
(B) All strains are on the same genetic background of BALB/c (C), an important precondition for intercrossing the various transgenes without jeopardizing crucial practical issues of this project, such as the ability to adoptively transfer fully transformed tumor cells or premalignant B-lineage cells from transgenic mice. We hypothesize that strain C.IL6/BCL2/AID^-/- mice will develop IgM⁺ WM-like tumors.

Project 3 – Role of interleukine-6 in multiple myeloma

The ongoing research project, entitled "Preclinical validation of IL-6 for translational myeloma research," seeks to address outstanding questions on the biology of IL-6 in order to facilitate ongoing efforts to target IL-6 more effectively in patients with myeloma (Fig. 3). Using transgenic mouse models of IL-6-dependent plasmacytoma (PCT) as the principal experimental model system, we will: rigorously evaluate the biological significance of IL-6 in Myc-driven PCT development; weigh the relative contributions of autocrine and paracrine sources of IL-6 to plasma-cell tumor development; and assess the role that an IL-6 signaling pathway that has been largely neglected in myeloma research – i.e., IL-6 trans-signaling – plays in plasma-cell neoplasia in mice. Taking advantage of a set of newly developed transgenic BALB/c (C) mouse strains available only at The University of Iowa Carver College of Medicine, we are pursuing three important, closely interrelated, specific research aims.

Aim 1 is to determine the global significance of IL-6 in Myc-driven PCT in mice. The rationale for this aim includes findings that suggest IL-6 may be dispensable for plasma-cell tumors (i.e., that pathway activity can be maintained in the absence of an IL-6/IL-6R [IL-6 receptor] interaction). The constitutive activation of Stat3 in Abl/Myc retrovirus-induced PCT in mice and activated B-cell like diffuse large B-cell lymphoma in humans provides a key example to that end. Aim 2 is to evaluate the relative importance of autocrine versus paracrine IL-6 in Myc-driven PCT in mice. The rationale for this aim includes findings suggesting that autocrine IL-6 is important for carcinomas (such as lung and breast cancers), whereas paracrine IL-6 is critical for blood cancers (including lymphoma and myeloma). However, the evidence for the latter is largely circumstantial, and the significance of paracrine IL-6 for malignant plasma-cell transformation has never been unequivocally proven in a genetic study. Aim 3 is to assess the role of IL-6 trans-signaling in Myc-driven PCT in mice. The rationale for this aim includes recent findings implicating IL-6 trans-signaling in inflammation-dependent solid and hematopoietic cancers in humans and mice and, more specifically, findings indicating that IL-6 trans-signaling plays a key role in the development of plasma-cell hyperplasia in mice.

Figure 3: Targeting IL-6 for improved outcome of MM. The scheme depicts novel approaches to inhibit IL-6 signaling in myeloma cells (bottom) and/or bone marrow stroma cells (top): IL-6, red squares; IL-6R/gp80, Y-shaped symbol; gp130, blue line). Numbered yellow circles indicate active research areas, which can be categorized as follows:
(1) Antibodies to, or antagonists of, IL-6R, including Sant7 (a modified human IL-6 that binds to gp80 with higher affinity than normal IL-6 does; however, because Sant7 does not recruit gp130 to the IL-6R, it blocks downstream IL-6 signaling), MRA (humanized mouse antibody to IL-6R; ACTEMBRA 200 [Chugai Pharmaceuticals]), ERBA (IL6R antagonist), and NRI/Tocilizumab.
(2) Antibodies to IL-6, including mouse monoclonal antibody BE-8 and chimeric antibody CNTO 328 (Centocor), which is currently being tested in a phase-2 clinical trial with and without Velcade.
(3) Inhibitors of IL-6 trans-signaling, including sgp130-Fc (R&D Systems), which captures circulating IL-6 and thus prevents it from binding to membrane-bound IL-6R.
(4) Inhibitors of gp130, such as gp130-targeting peptides.
(5) Small-molecule inhibitors, including Atiprimod (Callisto).