ALBERT

Description: CpG ODN are being actively investigated as cancer vaccine adjuvants because they mature plasmacytoid dendritic cells (pDC) into potent antigen-presenting cells. In addition, TLR ligands induce a broad range of other immunologic effects in pDC including the secretion of interferon α (IFNa) and chemokines which alter lymphocyte migration. Whether these CpG-ODN driven TLR ligand signals enhance antigen specific Immunity and/or trafficking In humans Is presently unknown. We evaluated the efficacy of the CpG-ODN, 1018-ISS, as a vaccine adjuvant given with GM-CSF to induce T cell immunity in humans to the tumor antigen hTERT. Seventeen patients with advanced solid tumors were treated with 6 cycles of GM-CSF (x 3d), CpG-ODN (escalating from 3mg - 100mg × 1d) followed by a peptide vaccine (a CD8 epitope from hTERT), in a Phase I dose escalation study. Surprisingly, only one of seventeen patients showed a detectable hTERT-specific tetramer T cell response. However, the majority of patients developed marked peripheral blood (PB) lymphopenia after CpG-ODN. Time-course flow cytometry analysis of PB revealed that CD8, CD4, NK and B cell counts were all depressed immediately after CpG-ODN. The effect was transient, with normal counts returning after a week, suggesting that CpG-ODN induced alteration in cell migration rather than cell death. To find further evidence for altered migration we examined vaccine sites. Clinically, vaccine sites showed significant swelling/induration within hours of CpG-ODN administration, though none was dose-limiting. Immunohistochemistry of vaccine biopsies showed significant, perivascular accumulation of CD4 and CD8 T cells clustered around CD123+ pDC. Biopsies after CpG-ODN, but not after GM-CSF, showed a marked increase in expression of MxA, an interferon-inducible gene suggesting that the local activation of pDC with resultant IFNa secretion. qRT-PCR confirmed significant increases in a panel of IFNa-inducible genes in the PB after CpG-ODN, indicating a systemic effect of IFNa secretion. Lastly, we showed directly that CpG-ODN markedly increased the ability of purified pDC to induce T cell migration in an in vitro transwell assay, demonstrating that CpG-ODN stimulation of human pDC not only induces IFNa, but also other chemokines that are sufficient to chemoattract T cells. Our results show that CpG-ODN with GM-CSF may not be an effective adjuvant strategy for peptide tumor vaccines; but sequenced GM-CSF/CpG-ODN causes a chemokine response that effects T cell migration to the peripheral tissues. These findings suggest a role for CpG beyond that of a vaccine adjuvant as a mediator of lymphocyte migration, targeting immune responses to specific peripheral tissues. Therapeutic intratumoral GM-CSF/CpG-ODN injection could profoundly alter the local immunologic milieu, recruiting activated pDC and T cells to the tumor site, and tipping the balance towards an effective tumor-specific immune response.

Description: The malignant microenvironment of FL, including T cells, macrophages and follicular dendritic cells, contributes to disease pathogenesis and represents a rational therapeutic target. Gene expression profiling and immunohistochemistry studies have confirmed that degree and type of immune response in the malignant FL lymph node impacts clinical outcome. CpG oligonucleotides have pleotropic effects on both the innate and adaptive immune systems, including increased antigen expression, enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and shifting the immune response in a TH-1 direction. These agents therefore have significant potential to manipulate the immune response to FL, and alter the malignant microenvironment in a favorable way. We combined a CpG oligonucleotide (1018 ISS, 0.2 mg/kg sc weekly x 4 beginning day 8) with standard R in patients (pts) with relapsed/refractory, histologically confirmed FL, and comprehensively evaluated immunological changes following the combination. 22 pts (10 female; median age 55) with FL sensitive or naive to R were enrolled. FcγRIIIA polymorphism status was FF n=9, VF n=12, VV n=1. Treatment was well-tolerated with no significant adverse events attributable to therapy. Baseline and post therapy evaluations were performed on blood and the CpG injection sites in all pts. Compared to baseline, 7 pts demonstrated significant increases in ADCC after therapy using a conventional chromium-release assay. Circulating CD3-positive cells increased in 11 pts. Increased IFN-γrelease by Elispot occurred in 5 pts. 10 pts had significant increases in neutrophils, T cells, and macrophages in skin biopsies of CpG injection sites performed 24 hours post therapy compared with baseline. Pre- and post-biopsies of tumor tissue (including lymph nodes and bone marrow) were performed in 5 pts, and in all cases a significant infiltration of T cells (CD8 〉 CD4) and macrophages were observed following treatment. Clinical responses per Cheson criteria were observed in 45% of pts; median PFS was 18 months in responding pts, and 9 months for entire group; 12 pts have not yet required further therapy for FL. In conclusion, this group of pts with relapsed FL had favorable clinical outcome despite adverse prognostic factors. The addition of CpG to R had measurable and reproducible local and systemic immune effects in a substantial subset of pts. Unlike other cytokine/antibody combinations, this study is the first to histologically confirm perturbation of the local immune malignant microenvironment following systemic biological therapy of FL.

Description: Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q− syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q− syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q− syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q− syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q− syndrome.

Description: The study of hematopoiesis has been greatly facilitated by transplantation of blood cell populations into recipient animals. Efficient engraftment of donor cells generally requires ablation of the host hematopoietic system. The zebrafish has recently emerged as a developmental and genetic system to study hematopoiesis. To enable the study of hematopoietic stem cell (HSC) biology, immune cell function, and leukemogenesis in zebrafish, we have developed hematopoietic cell transplantation (HCT) into adult recipient animals conditioned by γ irradiation. Dose-response experiments showed that the minimum lethal dose (MLD) of 40 Gy led to the specific ablation of hematolymphoid cells and death by 14 days after irradiation. Sublethal irradiation doses of 20 Gy predominantly ablated lymphocytes and permitted transplantation of a lethal T-cell leukemia. Finally, transplantation of hematopoietic cells carrying transgenes yielding red fluorescent erythrocytes and green fluorescent leukocytes showed that HCT is sufficient to rescue the MLD, that recipient hematolymphoid tissues were repopulated by donor-derived cells, and that donor blood cell lineages can be independently visualized in living recipients. Together, these results establish transplantation assays to test for HSC function and oncogenic transformation in zebrafish.

Description: FLT3 receptor tyrosine kinase is expressed on lymphoid and myeloid progenitors in the hematopoietic system. Activating mutations in FLT3 have been identified in approximately 30% of patients with acute myelogenous leukemia, making it one of the most common mutations observed in this disease. Frequently, the mutation is an in-frame internal tandem duplication (ITD) in the juxtamembrane region that results in constitutive activation of FLT3, and confers interleukin-3 (IL-3)–independent growth to Ba/F3 and 32D cells. FLT3-ITD mutants were cloned from primary human leukemia samples and assayed for transformation of primary hematopoietic cells using a murine bone marrow transplantation assay. FLT3-ITDs induced an oligoclonal myeloproliferative disorder in mice, characterized by splenomegaly and leukocytosis. The myeloproliferative phenotype, which was associated with extramedullary hematopoiesis in the spleen and liver, was confirmed by histopathologic and flow cytometric analysis. The disease latency of 40 to 60 days with FLT3-ITDs contrasted with wild-type FLT3 and enhanced green fluorescent protein (EGFP) controls, which did not develop hematologic disease (〉 200 days). These results demonstrate that FLT3-ITD mutant proteins are sufficient to induce a myeloproliferative disorder, but are insufficient to recapitulate the AML phenotype observed in humans. Additional mutations that impair hematopoietic differentiation may be required for the development of FLT3-ITD–associated acute myeloid leukemias. This model system should be useful to assess the contribution of additional cooperating mutations and to evaluate specific FLT3 inhibitors in vivo.

Description: The transcription factor PU.1 is a key regulator of hematopoiesis and is essential for normal hematopoietic stem cell (HSC) function and myeloid and lymphoid differentiation. Knockout mice are embryonic or neonatal lethal and exhibit an early differentiation block at the transition from HSC to CMP and CLP. Mice with a deletion of a critical upstream regulatory element (URE) at −14kb has been deleted exhibited decreased PU.1 expression in HSC, progenitors and B cells to 20% of normal levels. In contrast, an increase of PU.1 expression was detected in early thymic progenitors. UREΔ/Δ animals exhibited a block in normal thymocyte differentiation and frequently progressed to fatal T cell lymphomas between 3 and 8 months of age. Tumors were clonal and transplantable into NOD-SCID recipients. Since aberrant PU.1 expression constituted the initiating mutation, we sought to identify cooperating mutations contributing to tumor development by surveying the genome for hypermethylated promoter regions using restriction landmark global scanning. Methylation patterns revealed a characteristic epigenetic footprint leading to selective tumor suppressor gene silencing in transformation of lymphoid but not myeloid UREΔ/Δ progenitors, since DNA methylation was significantly lower in the AML samples compared with lymphomas. One of the corresponding methylated genes that was silenced in all lymphomas analyzed but none of the AMLs was identified as Idb4 (encoding ID4, inhibitor of DNA binding 4). This observation is consistent with data describing ID4 as a strong tumor suppressor gene which is frequently silenced in neoplasms. The expression of Idb4 in 6 additional lymphomas was assessed by quantitative RT-PCR and showed a strong correlation between promoter hypermethylation and Idb4 downregulation. To test whether Idb4 silencing through promoter hypermethylation can be pharmacologically reversed, a UREΔ/Δ lymphoma cell line was established and treated with 5-aza-2′deoxycytidine. We found that Idb4 expression was restored to normal levels following increasing concentrations of this demethylating agent. Finally, we analyzed the functional relevance of the Idb4 downregulation by restoring Idb4 expression in the UREΔ/Δ tumor line. UREΔ/Δ lymphoma cells reexpressing Idb4 had a marked reduction in both the in vitro growth rate and the ability to cause tumors after transplantation into NOD/SCID recipient mice, indicating that Idb4 is a potent suppressor of UREΔ/Δ thymic tumorigenicity. Taken together, we provide evidence that epigenetic DNA modifications contribute significantly and selectively to lymphoid transformation in UREΔ/Δ mice, and suggest a role for Idb4 repression as a second event in PU.1-initiated transformation of lymphoid precursors.

Description: Immunostimulatory oligonucleotides containing nonmethylated CpG motifs (ISS) have multiple actions on adaptive and innate immune responses, including enhanced antigen presentation and costimulatory molecule expression, stimulation of dendritic cell maturation, and induction of cytokines that result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and Th1 immune responses. Through these mechanisms, we hypothesized ISS might be able to overcome R resistance. Our completed phase I study combining an ISS (1018 ISS, Dynavax Technologies) with R in patients (pts) with relapsed or refractory, advanced stage NHL, demonstrated that the combination is safe, and causes a dose-related increase in the expression of several interferon (IFN)-inducible genes.(Blood 105:489). We have subsequently enrolled 16 pts (median age 60; 9 female) with R-naïve or sensitive FL in a phase II trial to further investigate the efficacy and biological activity of this rational combination. Pts received 4 injections of 1018 ISS (0.2 mg/kg sc) at weekly intervals, beginning at the time of the second of 4 weekly standard R infusions. No grade III/IV toxicities occurred. Genotyping of 13 evaluable pts for the FcγRIII-A 158 polymorphism revealed VF (n=6) and FF (n=7). Flow cytometry of peripheral blood demonstrated a significant expansion of CD3+ cells in 9 pts following 4 weeks of 1018 ISS. When tested with allogeneic stimulation using an EBV transformed B cell line (Mann) in ELISPOT assay, T cell secretion of IFN-γ was increased following 1018 ISS (mean count pre 1018 ISS: 362; mean count post 1018 ISS: 565; p = 0.05), whereas T cell secretion of IL-2 did not change, both consistent with an emerging Th1 immune response. Using a standard chromium release assay, 7/13 pts (54%) demonstrated increases in ADCC of peripheral blood mononuclear cells following ISS compared to baseline (effector:target ratio of 25:1); including 5 pts with FCγRIIIA genotype 158 FF. Skin biopsies were obtained in all patients at baseline, and following ISS therapy at the injection site and a distant site. Following 1018 ISS, 5 pts (42%) had significant skin infiltration with CD8+ lymphocytes; and 5 pts had increased numbers of CD68+ cells at injection sites. Biopsies of tumor were obtained at baseline, and post 1018 ISS treatment when possible, and evidence of CD8+ T cell infiltration within residual FL nodules was observed following R and 1018 ISS therapy. As of 08/05, at a median follow-up of 9 months, only 3 pts have experienced disease progression, one of whom subsequently converted to CRu without further therapeutic intervention. We conclude that the combination of 1018 ISS and rituximab is safe, and demonstrates significant local and systemic biological activity. Moreover, the demonstration of enhanced ADCC in pts with an FCγRIIIA 158 FF genotype (expected poor prognosis following R in pts with FL), the associated development of a Th1 response, and measurable alteration of the tumor microenvironment all suggest ISS could overcome both innate and acquired R resistance mechanisms in pts with FL.

Description: Tyrosine kinase fusion oncogenes that occur as a result of chromosomal translocations have been shown to activate proliferative and antiapoptotic pathways in leukemic cells, but the importance of autocrine and paracrine expression of hematopoietic cytokines in leukemia pathogenesis is not understood. Evidence that leukemic transformation may be, at least in part, cytokine dependent includes data from primary human leukemia cells, cell culture experiments, and murine models of leukemia. This report demonstrates that interleukin (IL)-3 plasma levels are elevated in myeloproliferative disease (MPD) caused by the TEL/tyrosine kinase fusions TEL/platelet-derived growth factor beta receptor (PDGFβR), TEL/Janus kinase 2 (JAK2), and TEL/neurotrophin-3 receptor (TRKC). Plasma granulocyte-macrophage colony-stimulating factor (GM-CSF) levels were elevated by TEL/PDGFβR and TEL/JAK2. However, all of the fusions tested efficiently induced MPD in mice genetically deficient for both GM-CSF and IL-3, demonstrating that these cytokines are not necessary for the development of disease in this model system. Furthermore, in experiments using normal marrow transduced with TEL/PDGFβR retrovirus mixed with marrow transduced with an enhanced green fluorescent protein (EGFP) retrovirus, the MPD induced in these mice demonstrated minimal stimulation of normal myelopoiesis by the TEL/PDGFβR-expressing cells. In contrast, recipients of mixed GM-CSF–transduced and EGFP-transduced marrow exhibited significant paracrine expansion of EGFP-expressing cells. Collectively, these data demonstrate that, although cytokine levels are elevated in murine bone marrow transplant models of leukemia using tyrosine kinase fusion oncogenes, GM-CSF and IL-3 are not required for myeloproliferation by any of the oncogenes tested.

Description: Waldenström macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Protein kinase Cβ (PKCβ) regulates cell survival and growth in many B-cell malignancies. In this study, we demonstrate up-regulation of PKCβ protein in WM using protein array techniques and immunohistochemistry. Enzastaurin, a PKCβ inhibitor, blocked PKCβ activity and induced a significant decrease of proliferation at 48 hours in WM cell lines (IC50, 2.5-10 μM). Similar effects were demonstrated in primary CD19+ WM cells, without cytotoxicity on peripheral blood mononuclear cells. In addition, enzastaurin overcame tumor cell growth induced by coculture of WM cells with bone marrow stromal cells. Enzastaurin induced dose-dependent apoptosis at 48 hours mediated via induction of caspase-3, caspase-8, caspase-9, and PARP cleavage. Enzastaurin inhibited Akt phosphorylation and Akt kinase activity, as well as downstream p-MARCKS and ribosomal p-S6. Furthermore, enzastaurin demonstrated additive cytotoxicity in combination with bortezomib, and synergistic cytotoxicity in combination with fludarabine. Finally, in an in vivo xenograft model of human WM, significant inhibition of tumor growth was observed in the enzastaurin-treated mice (P = .028). Our studies therefore show that enzastaurin has significant antitumor activity in WM both in vitro and in vivo, providing the framework for clinical trials to improve patient outcome in WM.