Bryan Ciccarelli

A novel orally active proteasome inhibitor ONX 0912 triggers in vitro and in vivo cytotoxicity in multiple myeloma

Bortezomib therapy has proven successful for the treatment of relapsed, relapsed/refractory, and newly diagnosed multiple myeloma (MM). At present, bortezomib is available as an intravenous injection, and its prolonged treatment is associated with toxicity and development of drug resistance. Here we show that the novel proteasome inhibitor ONX 0912, a tripeptide epoxyketone, inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies. The anti-MM activity of ONX-0912 is associated with activation of caspase-8, caspase-9, caspase-3, and poly(ADP) ribose polymerase, as well as inhibition of migration of MM cells and angiogenesis. ONX 0912, like bortezomib, predominantly inhibits chymotrypsin-like activity of the proteasome and is distinct from bortezomib in its chemical structure. Importantly, ONX 0912 is orally bioactive. In animal tumor model studies, ONX 0912 significantly reduced tumor progression and prolonged survival. Immununostaining of MM tumors from ONX 0912-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Finally, ONX 0912 enhances anti-MM activity of bortezomib, lenalidomide dexamethasone, or pan-histone deacetylase inhibitor. Taken together, our study provides the rationale for clinical protocols evaluating ONX 0912, either alone or in combination, to improve patient outcome in MM.

Increased Incidence of Transformation and Myelodysplasia/Acute Leukemia in Patients With Waldenström Macroglobulinemia Treated With Nucleoside Analogs

PURPOSE Nucleoside analogs (NAs) are considered as appropriate agents in the treatment of Waldenström macroglobulinemia (WM), a lymphoplasmacytic lymphoma. Sporadic reports on increased incidence of transformation to high-grade non-Hodgkins lymphoma and development of therapy-related myelodysplasia/acute leukemia (t-MDS/AML) among patients with WM treated with NAs prompted us to examine the incidence of such events in a large population of patients with WM. PATIENTS AND METHODS We examined the incidence of these events in 439 patients with WM, 193 and 136 of whom were previously treated with and without an NA, respectively, and 110 of whom had similar long-term follow-up without treatment. The median follow-up for all patients was 5 years. RESULTS Overall, 12 patients (6.2%) either developed transformation (n = 9; 4.7%) or developed t-MDS/AML (n = 3; 1.6%) among NA-treated patients, compared with one patient (0.4%) who developed transformation in the non-NA treated group (P < .001); no such events occurred among untreated patients. Transformation and t-MDS/AML occurred at a median of 5 years from onset of NA therapy. The median survival of NA-treated patients who developed transformation did not differ from other NA-treated patients as a result of effective salvage treatment used for transformed disease. However, all NA-treated patients who developed t-MDS/AML died at a median of 5 months. CONCLUSION These data demonstrate an increased incidence of disease transformation to high-grade NHL and the development of t-MDS/AML among patients with WM treated with NAs.

Thalidomide and rituximab in Waldenstrom macroglobulinemia.

Thalidomide enhances rituximab-mediated, antibody-dependent, cell-mediated cytotoxicity. We therefore conducted a phase 2 study using thalidomide and rituximab in symptomatic Waldenstrom macroglobulinemia (WM) patients naive to either agent. Intended therapy consisted of daily thalidomide (200 mg for 2 weeks, then 400 mg for 50 weeks) and rituximab (375 mg/m(2) per week) dosed on weeks 2 to 5 and 13 to 16. Twenty-five patients were enrolled, 20 of whom were untreated. Responses were complete response (n = 1), partial response (n = 15), and major response (n = 2), for overall and major response rate of 72% and 64%, respectively, on an intent-to-treat basis. Median serum IgM decreased from 3670 to 1590 mg/dL (P < .001), whereas median hematocrit rose from 33.0% to 37.6% (P = .004) at best response. Median time to progression for responders was 38 months. Peripheral neuropathy to thalidomide was the most common adverse event. Among 11 patients experiencing grade 2 or greater neuropathy, 10 resolved to grade 1 or less at a median of 6.7 months. Thalidomide in combination with rituximab is active and produces long-term responses in WM. Lower doses of thalidomide (ie, <or= 200 mg/day) should be considered given the high frequency of treatment-related neuropathy in this patient population. This trial is registered at www.clinicaltrials.gov as #NCT00142116.

Combination of novel proteasome inhibitor NPI-0052 and lenalidomide trigger in vitro and in vivo synergistic cytotoxicity in multiple myeloma

Our recent study demonstrated that a novel proteasome inhibitor NPI-0052 is distinct from bortezomib (Velcade) and, importantly, triggers apoptosis in multiple myeloma (MM) cells resistant to bortezomib. Here we demonstrate that combining NPI-0052 and lenalidomide (Revlimid) induces synergistic anti-MM activity in vitro using MM-cell lines or patient MM cells. NPI-0052 plus lenalidomide-induced apoptosis is associated with (1) activation of caspase-8, caspase-9, caspase-12, caspase-3, and poly(ADP) ribose polymerase; (2) activation of BH-3 protein BIM; (3) translocation of BIM to endoplasmic reticulum; (4) inhibition of migration of MM cells and angiogenesis; and (5) suppression of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities. Importantly, blockade of BIM using siRNA significantly abrogates NPI-0052 plus lenalidomide-induced apoptosis. Furthermore, studies using biochemical inhibitors of caspase-8 versus caspase-9 demonstrate that NPI-0052 plus lenalidomide-triggered apoptosis is primarily dependent on caspase-8 signaling. In animal tumor model studies, low-dose combination of NPI-0052 and lenalidomide is well tolerated, significantly inhibits tumor growth, and prolongs survival. Taken together, our study provides the preclinical rationale for clinical protocols evaluating lenalidomide together with NPI-0052 to improve patient outcome in MM.

miR-30-5p Functions as a Tumor Suppressor and Novel Therapeutic Tool by Targeting the Oncogenic Wnt/β-Catenin/BCL9 Pathway

Wnt/β-catenin signaling underlies the pathogenesis of a broad range of human cancers, including the deadly plasma cell cancer multiple myeloma. In this study, we report that downregulation of the tumor suppressor microRNA miR-30-5p is a frequent pathogenetic event in multiple myeloma. Evidence was developed that miR-30-5p downregulation occurs as a result of interaction between multiple myeloma cells and bone marrow stromal cells, which in turn enhances expression of BCL9, a transcriptional coactivator of the Wnt signaling pathway known to promote multiple myeloma cell proliferation, survival, migration, drug resistance, and formation of multiple myeloma cancer stem cells. The potential for clinical translation of strategies to re-express miR-30-5p as a therapeutic approach was further encouraged by the capacity of miR-30c and miR-30 mix to reduce tumor burden and metastatic potential in vivo in three murine xenograft models of human multiple myeloma without adversely affecting associated bone disease. Together, our findings offer a preclinical rationale to explore miR-30-5p delivery as an effective therapeutic strategy to eradicate multiple myeloma cells in vivo.

CD27-CD70 interactions in the pathogenesis of Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is a B-cell malignancy characterized by an IgM monoclonal gammopathy and bone marrow (BM) infiltration with lymphoplasmacytic cells (LPCs). Excess mast cells (MCs) are commonly present in WM, and provide growth and survival signals to LPCs through several TNF family ligands (CD40L, a proliferation-inducing ligand [APRIL], and B-lymphocyte stimulator factor [BLYS]). As part of these studies, we demonstrated that WM LPCs secrete soluble CD27 (sCD27), which is elevated in patients with WM (P < .001 vs healthy donors), and serves as a faithful marker of disease. Importantly, sCD27 stimulated expression of CD40L on 10 of 10 BM MC samples and APRIL on 4 of 10 BM MC samples obtained from patients with WM as well as on LAD2 MCs. Moreover, the SGN-70 humanized monoclonal antibody, which binds to CD70 (the receptor-ligand partner of CD27), abrogated sCD27 mediated up-regulation of CD40L and APRIL on WM MCs. Last, treatment of severe combined immunodeficiency-human (SCID-hu) mice with established WM using the SGN-70 antibody blocked disease progression in 12 of 12 mice, whereas disease progressed in all 5 untreated mice. The results of these studies demonstrate a functional role for sCD27 in WM pathogenesis, along with its utility as a surrogate marker of disease and a target in the treatment of WM.

IgA and IgG hypogammaglobulinemia in Waldenström’s macroglobulinemia

Background Hypogammaglobulinemia is common in Waldenström’s macroglobulinemia. The etiology of this finding remains unclear, but it has been speculated to be based on tumor-induced suppression of the ‘uninvolved’ immunoglobulin production Design and Methods We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 untreated patients with Waldenström’s macroglobulinemia and investigated the associated clinicopathological findings and impact of therapy. We also sequenced eight genes (AICDA, BTK, CD40, CD154, NEMO, TACI, SH2D1A, UNG) implicated in immunoglobulin deficiency in 19 Waldenström’s macroglobulinemia patients with IgA and/or IgG hypogammaglobulinemia. Results At baseline 63.3%, 58.0% and 49.3% of the 207 patients had abnormally low serum levels of IgA, IgG, or both. No association between IgA and IgG hypogammaglobulinemia and disease burden, serum IgM levels, β2-microglobulin, International Prognostic Scoring System score, or incidence of recurrent infections was observed, although the presence of adenopathy and/or splenomegaly was associated with a lower incidence of hypogammaglobulinemia. Lower IgA and IgG levels were associated with disease progression in patients managed with a ‘watch and wait’ strategy. IgA and/or IgG levels remained abnormally low despite response to treatment, including complete remissions. A missense mutation in the highly conserved catalytic site of UNG was observed in a patient with hypogammaglobulinemia, warranting further study of this pathway in Waldenström’s macroglobulinemia. Conclusions IgA and IgG hypogammaglobulinemia is common in Waldenström’s macroglobulinemia and persists despite therapeutic intervention and response. IgA and IgG hypogammaglobulinemia does not predict the risk of recurrent infections in patients with Waldenström’s macroglobulinemia, although lower levels of serum IgA and IgG are associated with disease progression in Waldenström’s macroglobulinemia patients being managed with a ‘watch and wait’ strategy.

Hepcidin Is Produced by Lymphoplasmacytic Cells and Is Associated With Anemia in Waldenström's Macroglobulinemia

Waldenströms macroglobulinemia (WM) patients often present with anemia as their primary disease manifestation that may be related to hepcidin, an important regulator of iron homeostasis. We therefore determined hepcidin levels in 53 WM patients, and 20 age-matched healthy patient donors by hepcidin-25 ELISA. Serum hepcidin levels were elevated in WM patients versus healthy patients (P=.04), and correlated with BM disease involvement (P=.004), beta-2-microglobulin levels (P=.029), and inversely with hemoglobin (P=.05). No correlation with serum iron indices was observed, though in patients with high hepcidin levels, increased iron deposition in bone marrow macrophages was observed. Importantly, hepcidin transcripts and protein were produced by primary WM cells. Hepcidin levels correlated with serum IL-6 (P<.001) and C-Reactive Protein (P=.033) levels. The results of this study implicate hepcidin as a contributor to anemia in WM, and suggest that an iron re-utilization defect accompanies hepcidin overproduction leading to its sequestration in WM patients.

The HMG‐CoA inhibitor, simvastatin, triggers in vitro anti‐tumour effect and decreases IgM secretion in Waldenstrom macroglobulinaemia

Waldenstrom macroglobulinaemia (WM) is an incurable lymphoplasmacytic lymphoma with secretion of serum monoclonal immunoglobulin M (IgM). We previously showed that patients receiving cholesterol‐lowering statins, had the lowest IgM value in a large cohort of patients with WM. Simvastatin, a 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase inhibitor, induced inhibition of proliferation, cytotoxic effect and apoptosis in IgM secreting cell lines as well as in primary CD19+ WM cells. Interestingly, those effects were reversed by addition of mevalonate and geranylgeranylpyrophosphate, demonstrating that simvastatin inhibited cell growth, survival and IgM secretion on BCWM.1 WM cells by inhibition of geranylgeranylated proteins. Furthermore, simvastatin overcame tumour cell growth induced by co‐culture of WM cells with bone‐marrow stromal cells. Simvastatin also decreased IgM secretion by BCWM.1 cells at an early time‐point that had not affected cell survival. Simvastatin‐induced cytotoxicity was preceded by a decrease in Akt (protein kinase B, PKB) and extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinase (MAPK) pathways at 18 h. In addition, simvastatin induced an increase in stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) MAPK followed by caspase‐8, ‐9, ‐3 and poly(ADP‐ribose) polymerase (PARP) cleavages at 18 h, leading to apoptosis. Furthermore, simvastatin enhanced the cytotoxicity induced by bortezomib, fludarabine and dexamethasone. Our studies therefore support our earlier observation of statin‐mediated anti‐WM activity and provide the framework for future clinical trials testing simvastatin in WM.

Histone deacetylase inhibitors demonstrate significant preclinical activity as single agents, and in combination with bortezomib in Waldenström's macroglobulinemia.

We studied the role of histone deacetylase inhibitors in Waldenstroms macroglobulinemia (WM). Gene expression profiling of bone marrow CD19+ cells from 30 patients and 10 healthy donors showed overexpression of HDAC4, HDAC9, and Sirt5, with validation of HDAC9 overexpression by q-PCR in primary and BCWM.1 cells. Suberoylanilide hydroxamic acid, trichostatin A, panobinostat, and sirtinol demonstrated dose-dependent killing of BCWM.1 cells. TSA showed the greatest potency with IC50 of 70 nM. Importantly, HDAC9 activity was decreased following TSA treatment suggesting an essential role for this HDAC in WM therapy. The combination of bortezomib plus HDAC inhibitors resulted in at least additive tumor cell killing in BCWM.1 cells. TSA and bortezomib-induced apoptosis depended on a similar set of caspase activation, whereas their effect on cell cycle regulators was distinctly different. These results provided a framework for examining HDAC inhibitors as monotherapy, as well as combination therapy with bortezomib in WM.