June H. Myklebust

Anti-CD47 Antibody Synergizes with Rituximab to Promote Phagocytosis and Eradicate Non-Hodgkin Lymphoma

Monoclonal antibodies are standard therapeutics for several cancers including the anti-CD20 antibody rituximab for B cell non-Hodgkin lymphoma (NHL). Rituximab and other antibodies are not curative and must be combined with cytotoxic chemotherapy for clinical benefit. Here we report the eradication of human NHL solely with a monoclonal antibody therapy combining rituximab with a blocking anti-CD47 antibody. We identified increased expression of CD47 on human NHL cells and determined that higher CD47 expression independently predicted adverse clinical outcomes in multiple NHL subtypes. Blocking anti-CD47 antibodies preferentially enabled phagocytosis of NHL cells and synergized with rituximab. Treatment of human NHL-engrafted mice with anti-CD47 antibody reduced lymphoma burden and improved survival, while combination treatment with rituximab led to elimination of lymphoma and cure. These antibodies synergized through a mechanism combining Fc receptor (FcR)-dependent and FcR-independent stimulation of phagocytosis that might be applicable to many other cancers.

Hierarchy in somatic mutations arising during genomic evolution and progression of follicular lymphoma.

Follicular lymphoma (FL) is currently incurable using conventional chemotherapy or immunotherapy regimes, compelling new strategies. Advances in high-throughput sequencing technologies that can reveal oncogenic pathways have stimulated interest in tailoring therapies toward actionable somatic mutations. However, for mutation-directed therapies to be most effective, the mutations must be uniformly present in evolved tumor cells as well as in the self-renewing tumor-cell precursors. Here, we show striking intratumoral clonal diversity within FL tumors in the representation of mutations in the majority of genes as revealed by whole exome sequencing of subpopulations. This diversity captures a clonal hierarchy, resolved using immunoglobulin somatic mutations and IGH-BCL2 translocations as a frame of reference and by comparing diagnosis and relapse tumor pairs, allowing us to distinguish early versus late genetic eventsduring lymphomagenesis. We provide evidence that IGH-BCL2 translocations and CREBBP mutations are early events, whereas MLL2 and TNFRSF14 mutations probably represent late events during disease evolution. These observations provide insight into which of the genetic lesions represent suitable candidates for targeted therapies.

Cytotoxic CD4+ and CD8+ T lymphocytes, generated by mutant p21-ras (12Val) peptide vaccination of a patient, recognize 12Val-dependent nested epitopes present within the vaccine peptide and kill autologous tumour cells carrying this mutation.

Mutant p21‐ras proteins contain sequences that distinguish them from normal ras, and represent unique epitopes for T‐cell recognition of antigen‐bearing tumour cells. Here, we examined the capacity of CD4+ and CD8+ T cells, generated simultaneously by mutant‐ras‐peptide vaccination of a pancreatic‐adenocarcinoma patient, to recognize and lyse autologous tumour cells harbouring corresponding activated K‐ras epitopes. The patient was vaccinated with a purified 17mer ras peptide (KLVVVGAVGVGKSALTI), containing the Gly12 → Val substitution. Responding T cells were cloned following peptide stimulation, and CD4+ and CD8+ peptide‐specific cytotoxic T lymphocytes(CTL) were obtained. Transient pancreatic‐adenocarcinoma cell lines(CPE) were established in cell culture from malignant ascites of the patient, and were shown to harbour the same K‐ras mutation as found in the primary tumour. These cells were efficiently killed by the T‐cell clones and CD8+‐mediated cytotoxicity was HLA‐class‐I‐restricted, as demonstrated by inhibition of lysis by anti‐class‐I monoclonal antibodies. By employing as targets different class‐I‐matched tumour cell lines expressing a 12Val mutation, we were able to demonstrate HLA‐B35 as the restriction molecule, and further use of peptide‐sensitized EBV‐B cells as target cells identified VVVGAVGVG as the nonamer peptide responsible for CD8+‐T‐cell recognition. These data demonstrate that peptide vaccination with a single mutant p21‐ras‐derived peptide induces CD4+ and CD8+ CTL specific for nested epitopes, including the Gly → Val substitution at codon 12, and that both these T‐cell sub‐sets specifically recognize tumour cells harbouring the corresponding K‐ras mutation. Int. J. Cancer 72:784–790, 1997.

Characterization of early stages of human B cell development by gene expression profiling.

We have characterized several stages of normal human B cell development in adult bone marrow by gene expression profiling of hemopoietic stem cells, early B (E-B), pro-B, pre-B, and immature B cells, using RNA amplification and Lymphochip cDNA microarrays (n = 6). Hierarchical clustering of 758 differentially expressed genes clearly separated the five populations. We used gene sets to investigate the functional assignment of the differentially expressed genes. Genes involved in VDJ recombination as well as B lineage-associated transcription factors (TCF3 (E2A), EBF, BCL11A, and PAX5) were turned on in E-B cells, before acquisition of CD19. Several transcription factors with unknown roles in B lymphoid cells demonstrated interesting expression patterns, including ZCCHC7 and ZHX2. Compared with hemopoietic stem cells and pro-B cells, E-B cells had increased expression of 18 genes, and these included IGJ, IL1RAP, BCL2, and CD62L. In addition, E-B cells expressed T/NK lineage and myeloid-associated genes including CD2, NOTCH1, CD99, PECAM1, TNFSF13B, and MPO. Expression of key genes was confirmed at the protein level by FACS analysis. Several of these Ags were heterogeneously expressed, providing a basis for further subdivision of E-B cells. Altogether, these results provide new information regarding expression of genes in early stages of human B cell development.

B-cell signaling networks reveal a negative prognostic human lymphoma cell subset that emerges during tumor progression.

Human tumors contain populations of both cancerous and host immune cells whose malignant signaling interactions may define each patients disease trajectory. We used multiplexed phospho-flow cytometry to profile single cells within human follicular lymphoma tumors and discovered a subpopulation of lymphoma cells with impaired B cell antigen receptor (BCR) signaling. The abundance of BCR-insensitive cells in each tumor negatively correlated with overall patient survival. These lymphoma negative prognostic (LNP) cells increased as tumors relapsed following chemotherapy. Loss of antigen receptor expression did not explain the absence of BCR signaling in LNP tumor cells, and other signaling responses were intact in these cells. Furthermore, BCR signaling responses could be reactivated in LNP cells, indicating that BCR signaling is not missing but rather specifically suppressed. LNP cells were also associated with changes to signaling interactions in the tumor microenvironment. Lower IL-7 signaling in tumor infiltrating T cells was observed in tumors with high LNP cell counts. The strength of signaling through T cell mediator of B cell function CD40 also stratified patient survival, particularly for those whose tumors contained few LNP cells. Thus, analysis of cell–cell interactions in heterogeneous primary tumors using signaling network profiles can identify and mechanistically define new populations of rare and clinically significant cells. Both the existence of these LNP cells and their aberrant signaling profiles provide targets for new therapies for follicular lymphoma.

Expression of S100A4 by a variety of cell types present in the tumor microenvironment of human breast cancer

The S100A4 protein, which is involved in the metastasis process, is a member of the S100 superfamily of Ca‐binding proteins. Members of this family are multifunctional signaling proteins with dual extra and intracellular functions involved in the regulation of diverse cellular processes. Several studies have established a correlation between S100A4 protein expression and worse prognosis for patients with various malignancies including breast cancer. In this article, we have used specific antibodies in combination with immunohistochemistry (IHC) to identify the cell types that express S100A4 in human breast cancer biopsies obtained from high‐risk patients. IHC analysis of 68 tumor biopsies showed that the protein is expressed preferentially by various cell types present in the tumor microenvironment (macrophages, fibroblasts, activated lymphocytes), rather than by the tumor cells themselves. Moreover, we show that the protein is externalized by the stroma cells to the fluid that bathes the tumor microenvironment, where it is found in several forms that most likely correspond to charge variants. Using a specific ELISA test, we detected a significant higher concentration of S100A4 in the tumor interstitial fluid (TIF) as compared to their corresponding normal counterparts (NIF).

High PD-1 expression and suppressed cytokine signaling distinguish T cells infiltrating follicular lymphoma tumors from peripheral T cells.

Defects in T-cell function in patients with cancer might influence their capacity to mount efficient antitumor immune responses. Here, we identified highly reduced IL-4-, IL-10-, and IL-21-induced phosphorylation of STAT6 and STAT3 in tumor-infiltrating T cells (TILs) in follicular lymphoma (FL) tumors, contrasting other non-Hodgkin lymphoma TILs. By combining phospho-protein-specific flow cytometry with several T-cell markers, we identified that CD4(+)CD45RO(+)CD62L(-) FL TILs were largely nonresponsive to cytokines, in contrast to the corresponding autologous peripheral blood subset. We observed differential expression of the inhibitory receptor PD-1 in FL TILs and peripheral blood T cells. Furthermore, CD4(+)PD-1(hi) FL TILs, containing T(FH) and non-T(FH) cells, had lost their cytokine responsiveness, whereas PD-1 TILs had normal cytokine signaling. However, this phenomenon was not tumor specific, because tonsil T cells were similar to FL TILs. FL tumor cells were negative for PD-1 ligands, but PD-L1(+) histiocytes were found within the T cell-rich zone of the neoplastic follicles. Disruption of the microenvironment and in vitro culture of FL TILs could restore cytokine signaling in the PD-1(hi) subset. Because FL TILs in vivo probably receive suppressive signals through PD-1, this provides a rationale for testing PD-1 Ab in combination with immunotherapy in patients with FL.

Therapeutic effect of CD137 immunomodulation in lymphoma and its enhancement by Treg depletion

Despite the success of passive immunotherapy with monoclonal antibodies (mAbs), many lymphoma patients eventually relapse. Induction of an adaptive immune response may elicit active and long-lasting antitumor immunity, thereby preventing or delaying recurrence. Immunomodulating mAbs directed against immune cell targets can be used to enhance the immune response to achieve efficient antitumor immunity. Anti-CD137 agonistic mAb has demonstrated antitumor efficacy in various tumor models and has now entered clinical trials for the treatment of solid tumors. Here, we investigate the therapeutic potential of anti-CD137 mAb in lymphoma. We found that human primary lymphoma tumors are infiltrated with CD137+ T cells. We therefore hypothesized that lymphoma would be susceptible to treatment with anti-CD137 agonistic mAb. Using a mouse model, we demonstrate that anti-CD137 therapy has potent antilymphoma activity in vivo. The antitumor effect of anti-CD137 therapy was mediated by both natural killer (NK) and CD8 T cells and induced long-lasting immunity. Moreover, the antitumor activity of anti-CD137 mAb could be further enhanced by depletion of regulatory T cell (T(regs)). These results support the evaluation of anti-CD137 therapy in clinical trials for patients with lymphoma.

Activation of phosphatidylinositol 3-kinase is important for erythropoietin-induced erythropoiesis from CD34+ hematopoietic progenitor cells

OBJECTIVE Several transducing molecules, including JAK2, STAT5, MAP kinases, phosphatidylinositol 3-kinase (PI3K), phospholipase C-gamma1, and PKC are activated by interaction between erythropoietin (EPO) and the EPO receptor. The aim of this was to examine the relative involvement of PI3K in the development of glycophorin A (GPA)(+) erythroid cells from normal hematopoietic progenitor cells. MATERIALS AND METHODS CD34(+) hematopoietic progenitor cells or subpopulations obtained by FACS sorting were cultured in serum-free medium containing EPO with or without inhibitors for PI3K, p38, MEK, or PKC for various time periods before phenotypic analysis or detection of apoptosis by flow cytometry, cell cycle analysis, high-resolution tracking of cell division, Western blot analysis, or Akt kinase assay were performed. RESULTS The PI3K inhibitor LY294002 completely counteracted the EPO-induced proliferation of CD34(+) progenitor cells and CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 also highly suppressed the expanded erythropoiesis induced by the combined action of EPO and stem cell factor. The profound inhibitory effect of LY294002 on proliferation was caused by its induction of cell cycle arrest in the G(0)/G(1) phase of the cell cycle. Some cells acquired GPA expression before they went through cell division. This was completely blocked by LY294002, implying an inhibitory effect on maturation. In addition, LY294002 completely blocked the viability-enhancing effect of EPO in CD34(+)CD71(+)CD45RA(-) erythroid progenitors. LY294002 and various inhibitors of PKC completely suppressed the EPO-induced increase in the activity of Akt kinase, a direct downstream target of PI3K. CONCLUSIONS Our results point to an important role for PI3K in mediating EPO-induced survival, proliferation, and possibly maturation of early erythroid progenitors.

Activation of the CAMP signaling pathway increases apoptosis in human B‐precursor cells and is associated with downregulation of Mcl‐1 expression

During B‐ and T‐cell ontogeny, extensive apoptosis occurs at distinct stages of development. Agents that increase intracellular levels of cAMP induce apoptosis in thymocytes and mature B cells, prompting us to investigate the role of cAMP signaling in human CD10+ B‐precursor cells. We show for the first time that forskolin (which increases intracellular levels of cAMP) increases apoptosis in the CD10+ cells in a dose‐dependent manner (19%–94% with 0–1,000 μM forskolin after 48 hours incubation, IC50 = 150 μM). High levels of apoptosis were also obtained by exposing the cells to the cAMP analogue 8‐chlorophenylthio‐cAMP (8‐CPT‐cAMP). Specific involvement of cAMP‐dependent protein kinase (PKA) was demonstrated by the ability of a cAMP antagonist, Rp‐isomer of 8‐bromo‐adenosine‐ 3′, 5′‐ monophosphorothioate (Rp‐8‐Br‐cAMPS), to reverse the apoptosis increasing effect of the complementary cAMP agonist, Sp‐8‐Br‐cAMPS. Furthermore, we investigated the expression of Bcl‐2 family proteins. We found that treatment of the cells with forskolin or 8‐CPT‐cAMP for 48 hours resulted in a fourfold decline in the expression of Mcl‐1 (n = 6, P = 0.002) compared to control cells. The expression of Bcl‐2, Bcl‐xl, or Bax was largely unaffected. Mature peripheral blood B cells showed a smaller increase in the percentage of apoptotic cells in response to 8‐CPT‐cAMP (1.3‐fold, n = 6, P = 0.045) compared to B‐precursor cells, and a smaller decrease in Mcl‐1 levels (1.5‐fold, n = 4, P = 0.014). Taken together, these findings show that cAMP is important in the regulation of apoptosis in B‐progenitor and mature B cells and suggest that cAMP‐increased apoptosis could be mediated, at least in part, by a decrease in Mcl‐1 levels. J. Cell. Physiol. 180:71–80, 1999.