Chesi, Marta

Key Points The tumor microenvironment drives myeloma cell clonogenic growth and self-renewal through GDF15.

AbstractImmunomodulators are effective in controlling hematologic malignancy by initiating or reactivating host antitumor immunity to otherwise poorly immunogenic and immune suppressive cancers. We aimed to boost antitumor immunity in B-cell lymphoma by developing a tumor cell vaccine incorporating α-galactosylceramide (α-GalCer) that targets the immune adjuvant properties of NKT cells. In the Eμ-myc transgenic mouse model, single therapeutic vaccination of irradiated, α-GalCer–loaded autologous tumor cells was sufficient to significantly inhibit growth of established tumors and prolong survival. Vaccine-induced antilymphoma immunity required NKT cells, NK cells, and CD8 T cells, and early IL-12–dependent production of IFN-γ. CD4 T cells, gamma/delta T cells, and IL-18 were not critical. Vaccine treatment induced a large systemic spike of IFN-γ and transient peripheral expansion of both NKT cells and NK cells, the major sources of IFN-γ. Furthermore, this vaccine approach was assessed in several other hematopoietic tumor models and was also therapeutically effective against AML-ETO9a acute myeloid leukemia. Replacing α-GalCer with β-mannosylceramide resulted in prolonged protection against Eμ-myc lymphoma. Overall, our results demonstrate a potent immune adjuvant effect of NKT cell ligands in therapeutic anticancer vaccination against oncogene-driven lymphomas, and this work supports clinical investigation of NKT cell–based immunotherapy in patients with hematologic malignancies.

Abstract Introduction:The histone deacetylase inhibitor (HDACi) Panobinostat (LBH-589, Novartis Pharmaceutical Corp) belongs to a new class of chemotherapeutics with demonstrated anticancer activities in laboratory studies and within the clinic. Using pre-clinical models of leukemia and lymphoma we have demonstrated that HDACi mediate their therapeutic effects via the intrinsic apoptosis pathway. Here we investigated the ability of Panobinostat to induce apoptosis in human multiple myeloma (MM) cell lines, alone and in combination with agents that target pro-survival Bcl-2 proteins (ABT-737) and the extrinsic apoptosis pathway (rhTRAIL). Moreover, we have used the Vk*myc murine model of MM to demonstrate the therapeutic efficacy of Panobinostat and its ability to induce apoptosis in MM cells in vivo. Methods: JJN3, OPM-2, RPMI-8226 and U266 MM cell lines were incubated with Panobinostat, ABT-737 or rhTRAIL alone or in combination. Cell viability and cell cycle were assessed using flow cytometry. Histone acetylation was assessed by western blot. C57BL/6 mice (n = 32) bearing transplanted Vk*myc MM (2 months post-transplant) were treated with Panobinostat (25mg/kg, 4 days; 15mg/kg 5 days/week, 3 weeks) or vehicle (D5W). Serum paraprotein levels were assessed weekly in all mice. Mice were sacrificed at various time points to measure apoptotic and therapeutic effects. Results: Treatment of MM cell lines with Panobinostat resulted in time- and dose-dependent induction of apoptosis associated with G1 phase arrest and histone H3/H4 acetylation. Combinations of Panobinostat with ABT-737 or rhTRAIL synergised to mediate robust and rapid apoptosis in the human MM cells. Serum paraprotein in mice bearing Vk*myc MM cells was significantly reduced after 4 days of high dose (25mg/ml) Panobinostat compared with vehicle treated mice (p<0.01) and remained at this reduced level for the treatment period (≤60% reduction compared with vehicle treated mice). This was associated with a significant induction of apoptosis and a reduction in CD-138+ plasma cells within the bone marrow. Removal of treatment was associated with increased paraprotein levels, progression of extramedullary disease and death endpoints. Overall, Panobinostat induced a significant survival advantage compared with vehicle (median=36.5 vs. 16 days, p<0.01). Conclusions: These results suggest that the HDACi Panobinostat has single agent activity against MM cell lines in vitro and provides a significant therapeutic benefit to mice bearing syngeneic MM tumors. Moreover, combination therapies consisting of HDAC inhibitors with agents that target pro-survival Bcl-2 proteins or the TRAIL apoptosis pathway may provide a rational approach to the treatment of MM. Future studies will investigate the molecular events underpinning the effects mediated by Panobinostat, ABT-737 and TRAIL in the Vk*myc MM model. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B176.

Abstract Emerging evidence indicates that tumors can follow several evolutionary paths over a patient's disease course. With the use of serial genomic analysis of samples collected at different points during the disease course of 28 patients with multiple myeloma, we found that the genomes of standard-risk patients show few changes over time, whereas those of cytogenetically high-risk patients show significantly more changes over time. The results indicate the existence of 3 temporal tumor types, which can either be genetically stable, linearly evolving, or heterogeneous clonal mixtures with shifting predominant clones. A detailed analysis of one high-risk patient sampled at 7 time points over the entire disease course identified 2 competing subclones that alternate in a back and forth manner for dominance with therapy until one clone underwent a dramatic linear evolution. With the use of the Vk*MYC genetically engineered mouse model of myeloma we modeled this competition between subclones for predominance occurring spontaneously and with therapeutic selection.

Abstract The attrition rate for anticancer drugs entering clinical trials is unacceptably high. For multiple myeloma (MM), we postulate that this is because of preclinical models that overemphasize the antiproliferative activity of drugs, and clinical trials performed in refractory end-stage patients. We validate the Vk*MYC transgenic mouse as a faithful model to predict single-agent drug activity in MM with a positive predictive value of 67% (4 of 6) for clinical activity, and a negative predictive value of 86% (6 of 7) for clinical inactivity. We identify 4 novel agents that should be prioritized for evaluation in clinical trials. Transplantation of Vk*MYC tumor cells into congenic mice selected for a more aggressive disease that models end-stage drug-resistant MM and responds only to combinations of drugs with single-agent activity in untreated Vk*MYC MM. We predict that combinations of standard agents, histone deacetylase inhibitors, bromodomain inhibitors, and hypoxia-activated prodrugs will demonstrate efficacy in the treatment of relapsed MM.

Abstract Multiple myeloma (MM) is malignancy of isotype-switched, BM-localized plasma cells that frequently results in bone destruction, BM failure, and death. Important molecular subgroups are identified by three classes of recurrent immunoglobulin gene translocations and hyperdiploidy, both of which affect disease course. From a clinical standpoint, it is critical to identify MM patients carrying the t(4;14) translocation, which is present in 15% of myelomas and is associated with dysregulation of WHSC1/MMSET and often FGFR3. These patients should all receive bortezomib as part of their initial induction treatment because this has been shown to significantly prolong survival. In contrast, patients with translocations affecting the MAF family of transcription factors, del17p, or gene-expression profiling (GEP)–defined high-risk disease appear to have a worse prognosis that is not dramatically improved by any intervention. These patients should be enrolled in innovative clinical trials. The remaining patients with cyclin D translocations or hyperdiploidy do well with most therapies, and the goal should be to control disease while minimizing toxicity.

Abstract Waldenström's macroglobulinemia (WM) is a distinct clinicobiological entity defined as a B-cell neoplasm characterized by a lymphoplasmacytic infiltrate in bone marrow (BM) and IgM paraprotein production. Cytogenetic analyses were historically limited by difficulty in obtaining tumor metaphases, and the genetic basis of the disease remains poorly defined. Here, we performed a comprehensive analysis in 42 WM patients by using a high-resolution, array-based comparative genomic hybridization approach to unravel the genetic mechanisms associated with WM pathogenesis. Overall, 83% of cases have chromosomal abnormalities, with a median of three abnormalities per patient. Gain of 6p was the second most common abnormality (17%), and its presence was always concomitant with 6q loss. A minimal deleted region, including MIRN15A and MIRN16-1, was delineated on 13q14 in 10% of patients. Of interest, we reported biallelic deletions and/or inactivating mutations with uniparental disomy in tumor necrosis factor (TNF) receptor–associated factor 3 and TNFα-induced protein 3, two negative regulators of the nuclear factor-κB (NF-κB) signaling pathway. Furthermore, we confirmed the association between TRAF3 inactivation and increased transcriptional activity of NF-κB target genes. Mutational activation of the NF-κB pathway, which is normally activated by ligand receptor interactions within the BM microenvironment, highlights its biological importance, and suggests a therapeutic role for inhibitors of NF-κB pathway activation in the treatment of WM. [Cancer Res 2009;69(8):3579–88]

Abstract MMSET, identified by its fusion to the IgH locus in t(4;14)-associated multiple myeloma, possesses domains found within chromatin regulators, including the SET domain. MMSET protein is overexpressed and highly associated with chromatin in myeloma cell lines carrying t(4;14). MMSET possesses methyltransferase activity for core histone H3 lysine 4 and histone 4 lysine 20, whereas MMSET made in cells only modified H4. Segments of MMSET fused to the Gal4 DNA binding domain repressed transcription of a chromatin-embedded Gal4 reporter gene. MMSET-mediated repression was associated with increased H4K20 methylation gene and loss of histone acetylation. Consistent with this repressive activity, MMSET could form a complex with HDAC1 and HDAC2, mSin3a, and the histone demethylase LSD1, suggesting that it is a component of corepressor complexes. Furthermore, MMSET coexpression enhances HDAC1- and HDAC2-mediated repression in transcriptional reporter assays. Finally, shRNA-mediated knockdown of MMSET compromised viability of a myeloma cell line, suggesting a biologic role for the protein in malignant cell growth. Collectively, these data suggest that, by acting directly as a modifier of chromatin as well as through binding of other chromatin-modifying enzymes, MMSET influences gene expression and potentially acts as a pathogenic agent in multiple myeloma.