Purine nucleoside phosphorylase enables dual metabolic checkpoints that prevent T cell immunodeficiency and TLR7-associated autoimmunity.

Purine nucleoside phosphorylase (PNP) enables the breakdown and recycling of guanine nucleosides. PNP insufficiency in humans is paradoxically associated with both immunodeficiency and autoimmunity, but the mechanistic basis for these outcomes is incompletely understood. Here, we identify two immune lineage-dependent consequences of PNP inactivation dictated by distinct gene interactions. During T cell development, PNP inactivation is synthetically lethal with downregulation of the dNTP triphosphohydrolase SAMHD1. This interaction requires deoxycytidine kinase activity and is antagonized by microenvironmental deoxycytidine. In B lymphocytes and macrophages, PNP regulates Toll-like receptor 7 signaling by controlling the levels of its (deoxy)guanosine nucleoside ligands. Overriding this regulatory mechanism promotes germinal center formation in the absence of exogenous antigen and accelerates disease in a mouse model of autoimmunity. This work reveals that one purine metabolism gene protects against immunodeficiency and autoimmunity via independent mechanisms operating in distinct immune lineages and identifies PNP as a potentially novel metabolic immune checkpoint.

Establishment and Characterization of a Reliable Xenograft Model of Hodgkin Lymphoma Suitable for the Study of Tumor Origin and the Design of New Therapies.

To identify the cells responsible for the initiation and maintenance of Hodgkin lymphoma (HL) cells, we have characterized a subpopulation of HL cells grown in vitro and in vivo with the aim of establishing a reliable and robust animal model for HL. To validate our model, we challenged the tumor cells in vivo by injecting the alkylating histone-deacetylase inhibitor, EDO-S101, a salvage regimen for HL patients, into xenografted mice. Methodology: Blood lymphocytes from 50 HL patients and seven HL cell lines were used. Immunohistochemistry, flow cytometry, and cytogenetics analyses were performed. The in vitro and in vivo effects of EDO-S101 were assessed. Results: We have successfully determined conditions for in vitro amplification and characterization of the HL L428-c subline, containing a higher proportion of CD30-/CD15- cells than the parental L428 cell line. This subline displayed excellent clonogenic potential and reliable reproducibility upon xenografting into immunodeficient NOD-SCID-gamma (-/-)(NSG) mice. Using cell sorting, we demonstrate that CD30-/CD15- subpopulations can gain the phenotype of the L428-c cell line in vitro. Moreover, the human cells recovered from the seventh week after injection of L428-c cells into NSG mice were small cells characterized by a high frequency of CD30-/CD15- cells. Cytogenetic analysis demonstrated that they were diploid and showed high telomere instability and telomerase activity. Accordingly, chromosomal instability emerged, as shown by the formation of dicentric chromosomes, ring chromosomes, and breakage/fusion/bridge cycles. Similarly, high telomerase activity and telomere instability were detected in circulating lymphocytes from HL patients. The beneficial effect of the histone-deacetylase inhibitor EDO-S101 as an anti-tumor drug validated our animal model. Conclusion: Our HL animal model requires only 10³ cells and is characterized by a high survival/toxicity ratio and high reproducibility. Moreover, the cells that engraft in mice are characterized by a high frequency of small CD30-/CD15- cells exhibiting high telomerase activity and telomere dysfunction.

Alkylating histone deacetylase inhibitors may have therapeutic value in experimental myeloperoxidase-ANCA vasculitis.

Current therapies for treating antineutrophil cytoplasm autoantibody (ANCA)-associated vasculitis include cyclophosphamide and corticosteroids. Unfortunately, these agents are associated with severe adverse effects, despite inducing remission in most patients. Histone deacetylase inhibitors are effective in rodent models of inflammation and act synergistically with many pharmacological agents, including alkylating agents like cyclophosphamide. EDO-S101 is an alkylating fusion histone deacetylase inhibitor molecule combining the DNA alkylating effect of Bendamustine with a pan-histone deacetylase inhibitor, Vorinostat. Here we studied the effects of EDO-S101 in two established rodent models of ANCA-associated vasculitis: a passive mouse model of anti-myeloperoxidase IgG-induced glomerulonephritis and an active rat model of myeloperoxidase-ANCA microscopic polyangiitis. Although pretreatment with EDO-S101 reduced circulating leukocytes, it did not prevent the development of passive IgG-induced glomerulonephritis in mice. On the other hand, treatment in rats significantly reduced glomerulonephritis and lung hemorrhage. EDO-S101 also significantly depleted rat B and T cells, and induced DNA damage and apoptosis in proliferating human B cells, suggesting a selective effect on the adaptive immune response. Thus, EDO-S101 may have a role in treatment of ANCA-associated vasculitis, operating primarily through its effects on the adaptive immune response to the autoantigen myeloperoxidase.

Combined alkylation and histone deacetylase inhibition with EDO-S101 has significant therapeutic activity against brain tumors in preclinical models.

There is a clear unmet need for novel therapeutic agents for management of primary and secondary brain tumors. Novel therapeutic agents with excellent central nervous system (CNS) penetration and therapeutic activity are urgently needed. EDO-S101 is a novel alkylating and histone deacetylase inhibiting agent created by covalent fusion of bendamustine and vorinostat. We used murine models to perform CNS pharmacokinetic analysis and preclinical therapeutic evaluation of EDO-S101 for CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme. EDO-S101 has excellent CNS penetration of 13.8% and 16.5% by intravenous infusion and bolus administration respectively. It shows promising therapeutic activity against CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme with significant prolongation of survival compared to no-treatment controls. Therapeutic activity was higher with IV infusion compared to IV bolus. It should be evaluated further for therapeutic use in brain tumors.

The first-in-class alkylating deacetylase inhibitor molecule tinostamustine shows antitumor effects and is synergistic with radiotherapy in preclinical models of glioblastoma.

BACKGROUND: The use of alkylating agents such as temozolomide in association with radiotherapy (RT) is the therapeutic standard of glioblastoma (GBM). This regimen modestly prolongs overall survival, also if, in light of the still dismal prognosis, further improvements are desperately needed, especially in the patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors, in which the benefit of standard treatment is less. Tinostamustine (EDO-S101) is a first-in-class alkylating deacetylase inhibitor (AK-DACi) molecule that fuses the DNA damaging effect of bendamustine with the fully functional pan-histone deacetylase (HDAC) inhibitor, vorinostat, in a completely new chemical entity. METHODS: Tinostamustine has been tested in models of GBM by using 13 GBM cell lines and seven patient-derived GBM proliferating/stem cell lines in vitro. U87MG and U251MG (MGMT negative), as well as T98G (MGMT positive), were subcutaneously injected in nude mice, whereas luciferase positive U251MG cells and patient-derived GBM stem cell line (CSCs-5) were evaluated the orthotopic intra-brain in vivo experiments. RESULTS: We demonstrated that tinostamustine possesses stronger antiproliferative and pro-apoptotic effects than those observed for vorinostat and bendamustine alone and similar to their combination and irrespective of MGMT expression. In addition, we observed a stronger radio-sensitization of single treatment and temozolomide used as control due to reduced expression and increased time of disappearance of γH2AX indicative of reduced signal and DNA repair. This was associated with higher caspase-3 activation and reduction of RT-mediated autophagy. In vivo, tinostamustine increased time-to-progression (TTP) and this was additive/synergistic to RT. Tinostamustine had significant therapeutic activity with suppression of tumor growth and prolongation of DFS (disease-free survival) and OS (overall survival) in orthotopic intra-brain models that was superior to bendamustine, RT and temozolomide and showing stronger radio sensitivity. CONCLUSIONS: Our data suggest that tinostamustine deserves further investigation in patients with glioblastoma.

Strategy for Assessing New Drug Value in Orphan Diseases: An International Case Match Control Analysis of the PROPEL Study.

BACKGROUND: Although randomized studies are designed to assess overall survival (OS) benefit, the conduct of regulatory studies in patients with orphan diseases can be timely and costly without offering the same commercial return on the investment. The peripheral T-cell lymphomas (PTCL) represent a rare group of heterogeneous lymphoid malignancies with very poor prognosis. PROPEL was a pivotal phase II study that led to the accelerated approval of pralatrexate for patients with relapsed or refractory PTCL. METHODS: An international database of 859 patients was assembled from four institutions with an interest in PTCL, of which 386 were considered eligible for matching against the PROPEL criteria. Using a rigorous propensity score matching algorithm, a unique 1:1 case match of 80 patients was performed. RESULTS: The analysis demonstrated an OS benefit for the PROPEL population with a median OS of 4.07 and 15.24 months (hazard ratio = 0.432, 95% confidence interval = 0.298 to 0.626), respectively, for the control and PROPEL populations. Highly statistically significant improvements in OS were noted for the PROPEL population about the subtype of PTCL (save anaplastic large cell lymphoma) and all age groups, including the elderly (>65 years of age). For patients on PROPEL, there was a statistically significant prolongation in progression free survival compared with the line of prior therapy, including those with refractory disease. CONCLUSION: In the context of this case-match-control study, patients treated on PROPEL experienced an OS advantage compared with an international database of historical controls. This information can help inform critical decision-making regarding clinical studies in PTCL.

Challenges in Optimising the Successful Construction of Antibody Drug Conjugates in Cancer Therapy.

Although considerable progress has been made in the field of cancer chemotherapy, there remains a significant unmet medical need, with a requirement to move away from traditional cytotoxics and explore novel, smarter chemotherapeutic approaches. One such example of the smart chemotherapy approach is antibody-drug conjugates (ADCs), which consist of an antibody that binds selectively to a cancer antigen linked to a cytotoxic agent. When developing an ADC, it may be necessary to produce a variety of constructs to fully assess the optimal configuration for the molecule. By testing ADCs prepared using a range of cytotoxic agents, linkers, or different antibodies, it is possible to fully assess the optimal approach for this treatment modality before advancing to the clinic. Since the development and approval of first-generation ADCs, significant improvements in development technology have occurred. Here, we consider the advances made within the field of ADCs, focusing on the development of EDO-B278 and EDO-B776, both of which have demonstrated efficacy in preclinical testing. Although some limitations remain in this field of development, the potential reduction in toxicity offered by ADCs justifies the investment in research to find workable solutions that could ultimately provide patients with superior outcomes.

Correction: Mehrling, T.; et al. Challenges in Optimising the Successful Construction of Antibody Drug Conjugates in Cancer Therapy. Antibodies 2018, 7, 11.

The Conflict of Interest section of the published paper [1] has been updated as follows: [...].

Preclinical anti-myeloma activity of EDO-S101, a new bendamustine-derived molecule with added HDACi activity, through potent DNA damage induction and impairment of DNA repair.

BACKGROUND: Despite recent advances in the treatment of multiple myeloma (MM), the prognosis of most patients remains poor, and resistance to traditional and new drugs frequently occurs. EDO-S101 is a novel therapeutic agent conceived as the fusion of a histone deacetylase inhibitor radical to bendamustine, with the aim of potentiating its alkylating activity. METHODS: The efficacy of EDO-S101 was evaluated in vitro, ex vivo and in vivo, alone, and in combination with standard anti-myeloma agents. The underlying mechanisms of action were also evaluated on MM cell lines, patient samples, and different murine models. RESULTS: EDO-S101 displayed potent activity in vitro in MM cell lines (IC50 1.6-4.8 µM) and ex vivo in cells isolated from MM patients, which was higher than that of bendamustine and independent of the p53 status and previous melphalan resistance. This activity was confirmed in vivo, in a CB17-SCID murine plasmacytoma model and in de novo Vk*MYC mice, leading to a significant survival improvement in both models. In addition, EDO-S101 was the only drug with single-agent activity in the multidrug resistant Vk12653 murine model. Attending to its mechanism of action, the molecule showed both, a HDACi effect (demonstrated by α-tubulin and histone hyperacetylation) and a DNA-damaging effect (shown by an increase in γH2AX); the latter being again clearly more potent than that of bendamustine. Using a reporter plasmid integrated into the genome of some MM cell lines, we demonstrate that, apart from inducing a potent DNA damage, EDO-S101 specifically inhibited the double strand break repair by the homologous recombination pathway. Moreover, EDO-S101 treatment reduced the recruitment of repair proteins such as RAD51 to DNA-damage sites identified as γH2AX foci. Finally, EDO-S101 preclinically synergized with bortezomib, both in vitro and in vivo. CONCLUSION: These findings provide rationale for the clinical investigation of EDO-S101 in MM, either as a single agent or in combination with other anti-MM drugs, particularly proteasome inhibitors.

The Alkylating-HDAC Inhibition Fusion Principle: Taking Chemotherapy to the Next Level with the First in Class Molecule EDO-S101.

Chemotherapy may still be an essential component to treat cancer in combination with new targeted therapies. But chemotherapy needs to get smarter in order to make those combination regimens more effective and also more tolerable, particularly for an aging population. We describe the first time the synthesis and pharmacological testing of a fusion molecule comprising of the alkylator bendamustine and the HDAC-inhibitor vorinostat. The drug was designed to allow for the exploitation of both mechanisms of action simultaneously with the goal to provide a molecule with superior efficacy over the single agents. The pharmacological testing confirms the full functional capacity of both moieties and encouraging pharmacological data raises the hope that the drug may turn out to be a great addition to the armentarium of anticancer agents.

Is there hope to treat glioblastoma effectively?

Thomas Mehrling was appointed Managing Director of Mundipharma EDO GmbH, Basel, in January 2013 and brings extensive experience with more than 17 years in the industry to this role. During his career, he has held various senior positions in different companies across almost all functions in drug development and commercialization. Most recently, he held the position of International Director Oncology Strategy (2011-2013). From 2004 to 2011 he served as European Director Oncology at Mundipharma International Ltd. During his tenure the oncology business of the European Mundipharma network of independent associated companies was set up and two major products were launched in Europe, DepoCyte® and Levact® (Ribomustin®, Treanda®). He joined Mundipharma in 2000 as Head of Business Development. Prior to Mundipharma, he was Senior Vice President of the global CRO Medical Affairs at Staticon International, and prior to this he acted as Medical Leader at Takeda European R&D center. Dr. Mehrling is a certified Pharmacist with a PhD in pharmacology and a certified Physician trained in haemato-oncology. He obtained his PhD from Frankfurt University following work on developing a new 5-HT3 antagonist to treat nausea and vomiting and developed a particular interest in mechanisms of multidrug resistance into chemotherapy. Dr. Mehrling earned his MD degree through his work in the Department of Internal medicine at Frankfurt University (Hemato-oncology and Cardiology) where he worked for several years before starting his career in the pharmaceutical industry.

Detection and outcome of occult leptomeningeal disease in diffuse large B-cell lymphoma and Burkitt lymphoma.

The benefit of intrathecal therapy and systemic rituximab on the outcome of diffuse large B-cell lymphoma at risk of central nervous system disease is controversial. Furthermore, the effect of intrathecal treatment and rituximab in diffuse large B-cell and Burkitt lymphoma with occult leptomeningeal disease detected by flow cytometry at diagnosis is unknown. Untreated diffuse large B-cell (n=246) and Burkitt (n=80) lymphoma at clinical risk of central nervous system disease and having had pre-treatment cerebrospinal fluid were analyzed by flow cytometry and cytology. Spinal fluid involvement was detected by flow cytometry alone (occult) in 33 (13%) diffuse large B-cell and 9 (11%) Burkitt lymphoma patients, and detected by cytology in 11 (4.5%) and 5 (6%) patients, respectively. Diffuse large B-cell lymphoma with occult spinal fluid involvement had poorer survival (P=0.0001) and freedom from central nervous system relapse (P<0.0001) compared to negative cases. Burkitt lymphoma with occult spinal fluid involvement had an inferior freedom from central nervous system relapse (P=0.026) but not survival. The amount of intrathecal chemotherapy was quantitatively associated with survival in diffuse large B-cell lymphoma with (P=0.02) and without (P=0.001) occult spinal fluid involvement. However, progression of systemic disease and not control of central nervous system disease was the principal cause of treatment failure. In diffuse large B-cell lymphoma, systemic rituximab was associated with improved freedom from central nervous system relapse (P=0.003) but not with survival. Our results suggest that patients at risk of central nervous system disease should be evaluated by flow cytometry and that intrathecal prophylaxis/therapy is beneficial.

Forodesine has high antitumor activity in chronic lymphocytic leukemia and activates p53-independent mitochondrial apoptosis by induction of p73 and BIM.

Chronic lymphocytic leukemia (CLL) is an incurable disease derived from the monoclonal expansion of CD5(+) B lymphocytes. High expression levels of ZAP-70 or CD38 and deletions of 17p13 (TP53) and 11q22-q23 (ATM) are associated with poorer overall survival and shorter time to disease progression. DNA damage and p53 play a pivotal role in apoptosis induction in response to conventional chemotherapy, because deletions of ATM or p53 identify CLL patients with resistance to treatment. Forodesine is a transition-state inhibitor of the purine nucleoside phosphorylase with antileukemic activity. We show that forodesine is highly cytotoxic as single agent or in combination with bendamustine and rituximab in primary leukemic cells from CLL patients regardless of CD38/ZAP-70 expression and p53 or ATM deletion. Forodesine activates the mitochondrial apoptotic pathway by decreasing the levels of antiapoptotic MCL-1 protein and induction of proapoptotic BIM protein. Forodesine induces transcriptional up-regulation of p73, a p53-related protein able to overcome the resistance to apoptosis of CLL cells lacking functional p53. Remarkably, no differences in these apoptotic markers were observed based on p53 or ATM status. In conclusion, forodesine induces apoptosis of CLL cells bypassing the DNA-damage/ATM/p53 pathway and might represent a novel chemotherapeutic approach that deserves clinical investigation.