Francis Mussai

Neuroblastoma arginase activity creates an immunosuppressive microenvironment that impairs autologous and engineered immunity

Neuroblastoma is the most common extracranial solid tumor of childhood, and survival remains poor for patients with advanced disease. Novel immune therapies are currently in development, but clinical outcomes have not matched preclinical results. Here, we describe key mechanisms in which neuroblastoma inhibits the immune response. We show that murine and human neuroblastoma tumor cells suppress T-cell proliferation through increased arginase activity. Arginase II is the predominant isoform expressed and creates an arginine-deplete local and systemic microenvironment. Neuroblastoma arginase activity results in inhibition of myeloid cell activation and suppression of bone marrow CD34(+) progenitor proliferation. Finally, we demonstrate that the arginase activity of neuroblastoma impairs NY-ESO-1-specific T-cell receptor and GD2-specific chimeric antigen receptor-engineered T-cell proliferation and cytotoxicity. High arginase II expression correlates with poor survival for patients with neuroblastoma. The results support the hypothesis that neuroblastoma creates an arginase-dependent immunosuppressive microenvironment in both the tumor and blood that leads to impaired immunosurveillance and suboptimal efficacy of immunotherapeutic approaches.

Molecular basis and current strategies of therapeutic arginine depletion for cancer.

Renewed interest in the use of therapeutic enzymes combined with an improved knowledge of cancer cell metabolism, has led to the translation of several arginine depletion strategies into early phase clinical trials. Arginine auxotrophic tumors are reliant on extracellular arginine, due to the downregulation of arginosuccinate synthetase or ornithine transcarbamylase—key enzymes for intracellular arginine recycling. Engineered arginine catabolic enzymes such as recombinant human arginase (rh‐Arg1‐PEG) and arginine deiminase (ADI‐PEG) have demonstrated cytotoxicity against arginine auxotrophic tumors. In this review, we discuss the molecular events triggered by extracellular arginine depletion that contribute to tumor cell death.

Challenges of clinical trial design for targeted agents against pediatric leukemias.

The past 40 years have seen significant improvements in both event-free and overall survival for children with acute lymphoblastic and acute myeloid leukemia (ALL and AML, respectively). Serial national and international clinical trials have optimized the use of conventional chemotherapeutic drugs and, along with improvements in supportive care that have enabled the delivery of more intensive regimens, have been responsible for the major improvements in patient outcome seen over the past few decades. However, the benefits of dose intensification have likely now been maximized, and over the same period, the identification of new cytotoxic drugs has been limited. Therefore, challenges remain if survival is to be improved further. In pediatric ALL, 5-year-survival rates of over 85% have been achieved with risk-stratified therapy, but a notable minority of patients will still not be cured. In pediatric AML, different challenges remain. A slower improvement in overall survival has taken place in this patient population. Despite the obvious morphological heterogeneity of AML blasts, biological stratification is comparatively limited, and translation into risk-stratified therapeutic approaches has only best characterized by the use of retinoic acid for t(15;17)-positive AML. Even where prognostic markers have been identified, limited therapeutic options or multi-drug resistance of AML blasts has limited the impact on patient benefit. For both, the acute morbidities of current treatment remain significant and may be life-threatening alone. In addition, the Childhood Cancer Survivor Study (CCSS) highlighted many leukemia survivors develop one or more chronic medical conditions attributable to treatment (1, 2). As the biology of leukemogenesis has become better understood, key molecules and intracellular pathways have been identified that offer the possibility of targeting directly the leukemia cells while sparing normal cells. Consequently, there is now a drive to develop novel leukemia-specific or “targeted” therapies. These new classes of drugs will have mechanisms of action, toxicities, and therapeutic indices quite different from conventional cytotoxic drugs previously encountered, thus rendering current clinical trial methodologies inappropriate. Clinical trial methods will need to be adapted to accommodate these features of these new classes of drugs. This review will address the challenges and some of the techniques for developing clinical trials for targeted therapies.

The arginine metabolome in acute lymphoblastic leukemia can be targeted by the pegylated-recombinant arginase I BCT-100: Targeting arginine metabolism in ALL

Arginine is a semi‐essential amino acid that plays a key role in cell survival and proliferation in normal and malignant cells. BCT‐100, a pegylated (PEG) recombinant human arginase, can deplete arginine and starve malignant cells of the amino acid. Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood, yet for patients with high risk or relapsed disease prognosis remains poor. We show that BCT‐100 is cytotoxic to ALL blasts from patients in vitro by necrosis, and is synergistic in combination with dexamethasone. Against ALL xenografts, BCT‐100 leads to a reduction in ALL engraftment and a prolongation of survival. ALL blasts express the arginine transporter CAT‐1, yet the majority of blasts are arginine auxotrophic due to deficiency in either argininosuccinate synthase (ASS) or ornithine transcarbamylase (OTC). Although endogenous upregulation or retroviral transduced increases in ASS or OTC may promote ALL survival under moderately low arginine conditions, expression of these enzymes cannot prevent BCT‐100 cytotoxicity at arginine depleting doses. RNA‐sequencing of ALL blasts and supporting stromal cells treated with BCT‐100 identifies a number of candidate pathways which are altered in the presence of arginine depletion. Therefore, BCT‐100 provides a new clinically relevant therapeutic approach to target arginine metabolism in ALL.

Adjunctive treatment in juvenile nasopharyngeal angiofibroma: How should we approach recurrence?

A recent case of advanced, recurrent juvenile nasopharyngeal angiofibroma (JNA) at our institution has highlighted the limited evidence regarding adjunctive treatment. We present the case of a 10-year-old boy who is the first to undergo multiple-staged surgical resections alongside vincristine treatment. We performed a review of the literature analyzing the roles of radiation therapy, cytotoxic drugs, and novel targeted agents in JNA relapse. Small cohort studies suggest radiotherapy and flutamide are the most rational treatment options for residual and recurrent JNA. Our review highlights the need for further research into the management of primary and recurrent JNA.

The development of targeted new agents to improve the outcome for children with leukemia

ABSTRACT Introduction: Survival rates in pediatric leukemia have greatly improved in the last decades but still a substantial number of patients will relapse and die. New agents are necessary to overcome the limitations of conventional chemotherapy and hematopoietic stem cell transplantation and to reduce their undesirable long-term toxicities. The identification of driving molecular alterations of leukemogenesis in subsets of patients will allow the incorporation of new-targeted therapies. Areas covered: In this article the authors present a detailed review of the most recent advances in targeted therapies for pediatric leukemias. A comprehensive description of the biological background, adult data and early clinical trials in pediatrics is provided. Expert opinion: Clinical trials are the way to evaluate new agents in pediatric cancer. The development of new drugs in pediatric leukemia must be preceded by a solid biological rationale. Agents in development exploit all possible vulnerabilities of leukemic cells. Drugs targeting cell surface antigens, intracellular signaling pathways and cell cycle inhibitors or epigenetic regulators are most prominent. Major advances have occurred thanks to new developments in engineering leading to optimized molecules such as anti-CD19 bi-specific T-cell engagers (e.g. blinatumomab) and antibody-drug conjugates. The integration of new-targeted therapies in pediatric chemotherapy-based regimens will lead to improved outcomes.

Hypocalcemia in a patient with osteosarcoma and 22q11.2 deletion syndrome.

Hypocalcemia is a rare complication of osteosarcoma, having been described in only 4 reports. We present the case of a 16-year-old male with metastatic osteosarcoma of the right humerus who was found to have severe asymptomatic hypocalcemia. Cytogenetic analysis of peripheral blood revealed a microdeletion in band 22q11.2. Following amputation of the tumor-bearing extremity, the patients calcium levels increased, but did not normalize. These findings suggested that the etiology of his hypocalcemia was osteoblastic utilization of calcium by the tumor, exacerbated by 22q11.2 deletion syndrome.

Tumor-Derived GM-CSF Promotes Granulocyte Immunosuppression in Mesothelioma Patients

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated. Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition. Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR− granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients. Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859–72. ©2018 AACR.

Arginine auxotrophic gene signature in paediatric sarcomas and brain tumours provides a viable target for arginine depletion therapies

Paediatric sarcomas and brain tumours, remain cancers of significant unmet need, with a poor prognosis for patients with high risk disease or those who relapse, and significant morbidities from treatment for those that survive using standard treatment approaches. Novel treatment strategies, based on the underlying tumour biology, are needed to improve outcomes. Arginine is a semi-essential amino acid that is imported from the extracellular microenvironment or recycled from intracellular precursors through the combined expression of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) enzymes. The failure to express at least one of these recycling enzymes makes cells reliant on extracellular arginine – a state known as arginine auxotrophism. Here we show in large in silico patient cohorts that paediatric sarcomas and brain tumours express predominately the arginine transporter SLC7A1 and the arginine metabolising enzyme Arginase 2 (ARG2), but have low-absent expression of OTC. The arginine metabolic pathway correlated with the expression of genes associated with tumour pathogenesis, and overall survival in paediatric sarcomas. This gene signature of arginine auxotrophism indicates paediatric sarcomas and brain tumours are a viable target for therapeutic arginase drugs under current clinical trial development.Paediatric sarcomas and brain tumours, remain cancers of significant unmet need, with a poor prognosis for patients with high risk disease or those who relapse, and significant morbidities from treatment for those that survive using standard treatment approaches. Novel treatment strategies, based on the underlying tumour biology, are needed to improve outcomes. Arginine is a semi-essential amino acid that is imported from the extracellular microenvironment or recycled from intracellular precursors through the combined expression of the enzymes ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) enzymes. The failure to express at least one of these recycling enzymes makes cells reliant on extracellular arginine - a state known as arginine auxotrophism. Here we show in large in silico patient cohorts that paediatric sarcomas and brain tumours express predominately the arginine transporter SLC7A1 and the arginine metabolising enzyme Arginase 2 (ARG2), but have low-absent expression of OTC. The arginine metabolic pathway correlated with the expression of genes associated with tumour pathogenesis, and overall survival in paediatric sarcomas. This gene signature of arginine auxotrophism indicates paediatric sarcomas and brain tumours are a viable target for therapeutic arginase drugs under current clinical trial development.

Neuroblastoma arginine addiction subverts the anticancer immune response

ABSTRACT High-risk neuroblastoma continues to have a poor prognosis, stimulating the ongoing development of alternative immune-therapy approaches. Despite exciting preclinical results, clinical outcomes have been less clear. Recently we identified that neuroblastoma expresses Arginase II, depleting local and systemic arginine concentrations, and suppression of autologous and engineered T cell immunity.