Anti-CD38 antibody therapy: windows of opportunity yielded by the functional characteristics of the target molecule.

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

HER2-positive breast cancer cells resistant to trastuzumab and lapatinib lose reliance upon HER2 and are sensitive to the multitargeted kinase inhibitor sorafenib.

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.

A high percentage of skin melanoma cells expresses SPANX proteins.

The expression of SPANX (sperm protein associated with the nucleus in the X chromosome) gene family has been reported in many tumors, such as melanoma, myeloma, glioblastoma, breast carcinoma, ovarian cancer, testicular germ cell tumors, and hematological malignancies. However, no systematic approach has so far been devised to estimate the percentage of cancer cells expressing SPANX. This study was undertaken to quantify the expression of SPANX proteins in melanomas. The expression of SPANX proteins was evaluated by immunohistochemistry in normal skin (n = 12), melanomas (n = 21), and benign nevi (n = 10), using a polyclonal antibody raised in our laboratory. Seventeen of the 21 melanomas (80.9%) examined expressed SPANX proteins. A high percentage of their cells (49.0% +/- 5.5%) stained positively for SPANX proteins compared with no expression found in normal skin cells. Benign nevi had an intermediate number of cells expressing SPANX proteins (25% +/- 8.5%), which resulted significantly higher than normal skin cells and significantly lower than skin melanoma cells. In melanoma cells, the labeling was mostly nuclear, sometimes incomplete or limited to the perinuclear wall, even if cytoplasmic staining was also seen in SPANX-positive tumor cells. In contrast, the 5 of 10 SPANX-positive nevi had a clear nuclear localization of the signal. These data suggest that the SPANX protein family is expressed in the vast majority of the melanomas tested. The mechanism(s), which brings up SPANX gene expression and the role of these proteins are not known.

A non-canonical adenosinergic pathway led by CD38 in human melanoma cells induces suppression of T cell proliferation.

Nucleotide-metabolizing ectoenzymes are endowed with an extracellular catalytic domain, which is involved in regulating the extracellular nucleotide/nucleoside balance. The tumor microenvironment contains high levels of adenosine (ADO) generated by this enzymatic network, thus promoting tumor growth by inhibiting anti-tumor immune responses. ADO inhibition in melanoma murine models limits tumor metastases and restores anti-tumor immune responses. This work investigates the expression and function of ectoenzymes in primary human melanoma cell lines. All of latter cells expressed CD38, CD39, CD73, and CD203a/PC-1, and produced ADO from AMP and NAD(+ )T cell proliferation. Accordingly, phosphorylation of S6 ribosomal protein, p38 and Stat1 was lower in activated memory cells than in naïve CD4(+) T lymphocytes. Melanoma cells also inhibited proliferation of naïve, memory and -to a lesser extent- of effector CD8(+) T cells. These different inhibitory effects correlated with distinct patterns of expression of the ADO receptor A2a and A2b. These results show that primary human melanoma cell lines suppress in vitro T cell proliferation through an adenosinergic pathway in which CD38 and CD73 play a prominent role.

Unraveling the contribution of ectoenzymes to myeloma life and survival in the bone marrow niche.

The bone marrow provides a protected environment for generating a vast array of cell types. Bones are thus a dynamic source of structural components and soluble factors used either locally or at a distance from their site of production. We discuss the role of ectoenzymes in the bone niche where human myeloma grows. Selected ectoenzymes have been tested for their ability to promote production of substrates involved in signaling, synthesis of growth factors and hormones, and modulation of the immune response. Because of the difficulty of simultaneously tracking all these activities, we narrow our focus to events potentially influencing synthesis of adenosine (ADO), an important regulator of multiple biological functions, including local immunological tolerance. Our working hypothesis, to be discussed and partially tested herein, is that CD38, and likely BST1/CD157--both NAD(+) -consuming enzymes, are active in the myeloma niche and lead a discontinuous chain of ectoenzymes whose final products are exploited by the neoplastic plasma cell as part of its local survival strategy. Coadjuvant ectoenzymes include PC-1/CD203a, CD39, and CD73, which control the production of ADO. Results discussed here and from ongoing experiments indicate that the myeloma niche hosts the canonical, as well as alternative, pathways of ADO generation. Other possibilities are presented and discussed.

CD38 and bone marrow microenvironment.

This review summarizes the events ruled by CD38 shaping the bone marrow environment, recapitulating old and new aspects derived from the body of knowledge on the molecule. The disease models considered were myeloma and chronic lymphocytic leukemia (CLL). CD38 has been analyzed considering its twin function as receptor and enzyme, roles usually not considered in clinics, where it is used as a routine marker. Another aspect pertaining basic science concerns the role of the molecule as a member of an ectoenzyme network, potentially metabolizing soluble factors not yet analyzed (e.g., NAD+, ATP, NAM) or influencing hormone secretion (e.g., oxytocin). The last point is focused on the use of CD38 as a target of an antibody-mediated therapeutic approach in myeloma and CLL. A recent observation is that CD38 may run an escape circuit leading to the production of adenosine. The generation of local anergy may be blocked by using anti-CD38 antibodies. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

CD38 and CD157: a long journey from activation markers to multifunctional molecules.

CD38 (also known as T10) was identified in the late 1970s in the course of pioneering work carried out at the Dana-Farber Cancer Center (Boston, MA) that focused on the identification of surface molecules involved in antigen recognition. CD38 was initially found on thymocytes and T lymphocytes, but today we know that the molecule is found throughout the immune system, although its expression levels vary. Because of this, CD38 was considered an "activation marker," a term still popular in routine flow cytometry. This review summarizes the findings obtained from different approaches, which led to CD38 being re-defined as a multifunctional molecule. CD38 and its homologue CD157 (BST-1), contiguous gene duplicates on human chromosome 4 (4p15), are part of a gene family encoding products that modulate the social life of cells by means of bidirectional signals. Both CD38 and CD157 play dual roles as receptors and ectoenzymes, endowed with complex activities related to signaling and cell homeostasis. The structure-function analysis presented here is intended to give clinical scientists and flow cytometrists a background knowledge of these molecules. The link between CD38/CD157 and human diseases will be explored here in the context of chronic lymphocytic leukemia, myeloma and ovarian carcinoma, although other disease associations are also known. Thus CD38 and CD157 have evolved from simple leukocyte activation markers to multifunctional molecules involved in health and disease. Future tasks will be to explore their potential as targets for in vivo therapeutic interventions and as regulators of the immune response.

A CD38/CD203a/CD73 ectoenzymatic pathway independent of CD39 drives a novel adenosinergic loop in human T lymphocytes.

The tumor microenvironment is characterized by of high levels of extracellular nucleotides that are metabolized through the dynamic and sequential action of cell surface enzymes (ectoenzymes). These ectoenzymes operate according to their spatial arrangement, as part of (1) continuous (molecules on the same cell) or (2) discontinuous (molecules on different cells) pathways, the latter being facilitated by restricted cellular microenvironment. The outcome of this catabolic activity is an increase in the local concentration of adenosine, a nucleoside involved in the control of inflammation and immune responses. The aim of the work presented here was to demonstrate that a previously unexplored enzymatic pathway may be an alternate route to produce extracellular adenosine. Our data show that this new axis is driven by the nucleotide-metabolizing ectoenzymes CD38 (an NAD+ nucleosidase), the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1, also known as CD203a or PC-1) and the 5' ectonucleotidase (5'-NT) CD73, while bypassing the canonical catabolic pathway mediated by the nucleoside tri- and diphosphohydrolase (NTPDase) CD39. To determine the relative contributions of these cell surface enzymes to the production of adenosine, we exploited a human T-cell model allowing for the modular expression of the individual components of this alternative pathway upon activation and transfection. The biochemical analysis of the products of these ectoenzymes by high-performance liquid chromatography (HPLC) fully substantiated our working hypothesis. This newly characterized pathway may facilitate the emergence of an adaptive immune response in selected cellular contexts. Considering the role for extracellular adenosine in the regulation of inflammation and immunogenicity, this pathway could constitute a novel strategy of tumor evasion, implying that these enzymes may represent ideal targets for antibody-mediated therapy.

The hidden life of NAD+-consuming ectoenzymes in the endocrine system.

Ectoenzymes are a family of cell surface molecules whose catalytic domain lies in the extracellular region. A subset of this family, nucleotide-metabolizing ectoenzymes, are key components in the regulation of the extracellular balance between nucleotides (e.g. NAD(+) or ATP) and nucleosides (e.g. adenosine). Their substrates and products are signalling molecules that act by binding to specific receptors, triggering signals that regulate a variety of functions, ranging from the migration of immune cells, to synaptic transmission in the brain, to hormone/receptor interactions in the glands. Almost two decades of accumulated data indicate that these regulatory processes significantly affect the endocrine system, a tightly controlled information signal complex with clear evidence of fine regulation. Functional models discussed in this review include insulin secretion, bone modelling and the association between hormones and behaviour. The emerging pattern is one of a system operating as a scale-free network that hinges around hubs of key molecules, such as NAD(+) or ATP. The underlying natural link between nucleotides, ectoenzymes and the endocrine system is far from being clearly demonstrated. However, the body of evidence supporting the existence of such connection is growing exponentially. This review will try to read the available evidence in a hypothesis-oriented perspective, starting from the description of NAD(+) and of ecto- and endoenzymes involved in its metabolism.