Histone acetylation represents another important pathway in epigenetic gene regulation. Histone deacetylases (HDACs) are involved in transcription regulation and signal transduction of cells through modification of histones and other proteins. HDAC-specific inhibitors (Table 1) have anti-proliferative activity and several of them are being investigated clinically in other B-cell malignancies. Using gene expression profiling, we recently showed that HDACs are up-regulated in primary WM cells.

Table 1. Histone deacetylase family comprises of 18 members, which are sub-divided into four classes. Members and inhibitors of each class are included.

As part of these efforts, we sought to delineate the activity of HDAC class I, II, III inhibitors in WM by examining the activity of the HDAC inhibitors Vorinostat (Class I), Trichostatin A (Class II), and Sirtinol (Class III) as mono-therapy and in combination with Bortezomib in BCWM.1 WM cells. These studies demonstrated a dose-dependent increase in tumor cell killing by MTT assays, Annexin V and PI staining for all 3 classes of HDAC inhibitors with the following IC50 Vorinostat (3uM); Trichostatin A (60 uM); Sirtinol (5 uM) in BCWM.1 WM cells at 24 hours. Importantly, the combination of each of the 3 classes HDAC inhibitors with sub-lethal doses of the proteasome inhibitor Bortezomib resulted in synergistic tumor cell killing. In view of the above results, gene expression profiling and in vivo studies in SCID-hu mice were initiated to further define the activity of combined HDAC and proteosome inhibition in WM. The results of these studies were presented at the 5th International Workshop on WM (Sun et al, www.wmworkshop.org) and suggest a role for HDAC inhibitors, particularly in combination with Bortezomib, as novel treatment strategies for WM.

Studies into Endoplasmic Reticulum Stress in WM. The endoplasmic reticulum (ER) is the site where integral proteins and secretory proteins are folded into their tertiary structure, and multimeric proteins, such as immunoglobulins, are assembled. To survive under endoplasmic reticulum (ER) stress, eukaryotic cells have a self-protective mechanism against ER stress, termed the unfolded protein response (UPR). The specific induction of UPR genes enables cells to differentiate into professional secretory cells capable of tolerating the constitutive production of high amounts of ER-processed proteins, a process termed the "physiologic" UPR. The UPR maintains the quality of newly synthesized secretory and transmembrane proteins such as immunoglobulins, and is distinct from the "ER stress" or "terminal/proapoptotic" UPR, which is induced by nutrient deprivation or chemical agents that cause severe or prolonged ER stress, and triggers an apoptotic cascade called proapoptotic/terminal UPR. Several studies reveal a critical role for UPR activation for tumor cell resistance to hypoxia and tumor growth promotion, and suggest that the UPR may be an attractive target for antitumor modalities. It is therefore reasonable to assume that manipulation of ER stress might enhance the efficacy of chemotherapeutic drugs and provide new anticancer targets. So far, data support the potential of drugs that inhibit the normal functions and homeostasis of the ER in treatment of malignancies, and has lead to the development of heat shock protein 90 inhibitors. Interestingly, another novel agent, the proteasome inhibitor bortezomib, has been shown to target the ER function, by inducing components of the proapoptotic/terminal UPR. As part of these studies, we showed that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, an ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 µg/mL to 1 µg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrated that ER stress inducer synergized with other agents used in the treatment of WM. The results of this study were recently published (Leleu et al, Blood 2008). These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

Studies into CD70-CD27 interactions in WM. Recent studies published by us (Ho et al, BLOOD 2008) demonstrated an important role for sCD27-CD70 signaling among BM LPCs and MCs in WM, and demonstrate a novel mechanism of action for sCD27 as a regulator of 2 principal TNF family members (APRIL and CD40L) whose role as growth and survival factors has previously been established by us and others in WM and other B-cell malignancies (Figure 8).

Figure 8. Targeting WM cell growth and survival by use of agents that directly kill WM cells, by effecting Mast cell-WM cell support networks, as well as by stimulating anti-WM cell immunity.

Central to these studies was the demonstration that a monoclonal antibody (SGN-70) that bound to CD70, blocked sCD27 induced CD40L and APRIL upregulation on bone marrow mast cells taken from WM patients, and when used in a WM engrafted mice, blocked the progression of WM disease (Figure 9). These studies therefore support the investigation of SGN-70 as a novel therapy in WM patients.

Figure 9. WM engrafted SCID-hu mice treated with SGN-70 antibody (Group 2) showed inhibition of disease progression when compared to untreated mice (Group 1).

In addition to its functional role, these studies also suggested that sCD27 may serve as a faithful marker of disease in patients with WM, even among patients experiencing a rituximab related IgM flare or plasmapheresis (Ciccarelli et al, Clinical Lymphoma Myeloma 2009). Notably, our data also suggested that targeting CD70 and sCD27-CD70 interactions may produce important clinical benefits for patients with WM.

It remained unclear however from the above studies whether the high levels of sCD27 detected in patients with WM were the result of a malignant event, or part of a normal homeostatic mechanism for control of LPC expansion. Normally, CD27 is expressed on the cell surface of memory B cells from which WM is thought to derive. However, in WM, CD27 is heterogeneously expressed, and more often is absent on the cell surface of WM LPCs, and does not appear to occur as a result of a mutation event in CD27 gene itself (Ho et al, BLOOD 2008).

We therefore investigated the mechanisms by which CD27 is cleaved and released into the serum by examining the action of matrix metalloproteases (MMPs). These studies which are currently ongoing, suggest that MMP may in particular modulate CD27 release, and that a specific inhibitors may inhibit sCD27 release. Validation of these studies in SCID-hu mice are contemplated. Importantly, the use of MMP inhibitors may represent a novel class of therapeutics for WM by inhibiting sCD27 release.

The lack of cell-surface CD27 observed in this and other studies of LPCs in most patients with WM may also have important implications for the deregulation of LPC homeostasis in WM. Upon binding of CD70 to CD27, the pro-apoptotic adaptor proteins SIVA-1 and SIVA-2 bind to the intercellular domain of CD27 and trigger apoptosis via a caspase-dependent mitochondrial pathway. The loss of cell-surface expression of CD27, inhibition of CD70 signaling by tumor-released sCD27, or loss of SIVA-1 and SIVA-2 may represent pathways by which normal homeostasis of LPCs is lost in WM. As noted above, our sequencing studies did not detect any variants in CD27 to suggest that a solubilized form of this molecule resulted from mutation inducing truncation of this protein. However, it does remain possible that posttranscriptional or posttranslational events may still be shifted as an epigenetic malignant phenomenon resulting in the overproduction of sCD27.

Since our GEP studies demonstrated decreased SIVA levels, we undertook sequencing studies to determine if SIVA was mutated in WM patients. Gene sequence analysis of SIVA demonstrated a novel, previously unreported polymorphic site, which was observed in healthy donor specimens. Current studies are validating by real time PCR analysis SIVA levels in WM patient samples, and epigenetic modifications.

Studies into the Familial Predisposition of WM. As part of studies funded by the International Waldenstrom’s Macroglobulinemia Foundation (IWMF), we established a familial registry at DFCI in order to delineate familial predilections for WM by examining a large cohort of first and second degree family members of WM patients with and without familial histories for B-cell disorders. Approximately 450 individuals, representing 95 families including 41 families with a familial history of B-cell disorders have been enrolled. Prior medical history, complete blood counts, serum laboratories, immunoglobulin levels, serum immunofixation studies, peripheral blood and cheek cell DNA are being collected as part of this international study.

The interim results of this study (Hunter et al, Proceedings of the American Society of Oncology 2008) demonstrated an increased incidence of recurrent sinusitis, abnormally low IgA and IgG levels, elevated total IgM, and the presence of monoclonal gammopathy (predominantly IgM) among family members of WM patients with a familial history of B-cell disorders. Molecular studies to elucidate the underlying genetic basis for these observations are currently underway and include high density SNP arrays, and evaluation of patients and family members for mutations in common variable immunodeficiency (CVID) related genes (see below).

Studies into CVID related mutations in WM. Hypogammaglobulinemia of the "uninvolved" immunoglobulins is commonly observed in Waldenstrom’s macroglobulinemia (WM), and has often been attributed to disease-related suppression. However, there is a paucity of information related to the pathogenesis of hypogammaglobulinemia in these disorders. We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 patients with WM, and addressed the impact of therapy and response on IgA and IgG levels for 93 of these patients who required subsequent treatment. We also performed extensive sequence analysis of the promoter, all exonic, and flanking intronic regions from peripheral blood of 19 untreated WM patients who demonstrated IgA and/or IgG hypogammaglobulinemia for 8 genes often observed in common variable immunodeficiency disorders (CVID) and B cell deficiency i.e. AICDA, BTK, CD40, CD154, NEMO (IkBkG), TACI, SH2D1A, UNG. At baseline, 120/207 (58.0%), 131/207 (63.3%), and 102/196 (49.3%) patients had abnormally low levels of serum IgG (<700 mg/dL), IgA (<70 mg/dL), or both, respectively. No correlation between baseline bone marrow disease involvement and immunoglobulin levels was observed. With a median follow-up of 12 months following completion of therapy, IgA and/or IgG levels remained abnormally low for 92.1% and 87.3% of responding patients, respectively, including those who achieved a complete remission. Sequence analysis of the NEMO and CVID gene panel demonstrated intronic variation at position c.1056-6T>C (n=2) and a hemizygous missense mutation at codon 113 for NEMO (n=1), and a heterozygous missense mutation at codon 142 in UNG (n=1). The results of these studies demonstrate that IgA and IgG hypogammaglobulinemia is a constitutive feature of patients with WM, which neither correlates with, nor is impacted by disease burden, despite therapeutic intervention and response. The results also suggest that patients with WM may harbor sequence mutations, which may be a contributor to the pathogenesis and/or morbidity of WM. The results of these studies were presented at the 50th Annual Meeting of the American Society of Hematology (Treon et al, ASH 2008).