Scott W. Rowlinson

Systemic depletion of L-cyst(e)ine with cyst(e)inase increases reactive oxygen species and suppresses tumor growth

Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(−) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than non-malignant cells due to genetic alterations and abnormal growth and as a result, maintenance of the anti-oxidant glutathione (GSH) is essential for their survival and proliferation1–3. Under elevated ROS conditions endogenous l-Cysteine (l-Cys) production is insufficient for GSH synthesis, necessitating l-Cys uptake, predominantly in its disulfide form l-Cystine (CSSC) via the xCT(−) transporter. Here we show that administration of an engineered, pharmacologically optimized, human Cyst(e)inase enzyme mediates sustained depletion of the extracellular l-Cys and CSSC pool in mice and non-human primates, selectively causes cell cycle arrest and death (PI and Annexin-V staining) in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS, yet results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53−/− mice that develop disease resembling human chronic lymphocytic leukemia. The observation that enzyme-mediated depletion of the serum l-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods suggests that Cyst(e)inase represents a safe and effective therapeutic modality for inactivating anti-oxidant cellular responses in a wide range of malignancies4,5.

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency

Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency.

Oncogene-Selective Sensitivity to Synchronous Cell Death following Modulation of the Amino Acid Nutrient Cystine

Summary Cancer cells reprogram their metabolism, altering both uptake and utilization of extracellular nutrients. We individually depleted amino acid nutrients from isogenic cells expressing commonly activated oncogenes to identify correspondences between nutrient supply and viability. In HME (human mammary epithelial) cells, deprivation of cystine led to increased cell death in cells expressing an activated epidermal growth factor receptor (EGFR) mutant. Cell death occurred via synchronous ferroptosis, with generation of reactive oxygen species (ROS). Hydrogen peroxide promoted cell death, as both catalase and inhibition of NADPH oxidase 4 (NOX4) blocked ferroptosis. Blockade of EGFR or mitogen-activated protein kinase (MAPK) signaling similarly protected cells from ferroptosis, whereas treatment of xenografts derived from EGFR mutant non-small-cell lung cancer (NSCLC) with a cystine-depleting enzyme inhibited tumor growth in mice. Collectively, our results identify a potentially exploitable sensitization of some EGFR/MAPK-driven tumors to ferroptosis following cystine depletion.

Abstract 1042: Development of AEB1102, an engineered human arginase 1 for patients with solid tumors

Introduction: Tumor dependence on specific amino acids for survival and proliferation is well recognized and has been exploited effectively with the use of Asparaginase for the treatment of Acute Lymphoblastic Leukemia. Decades of research have led to an understanding of tumor L-Arginine (L-Arg) dependence, with functional expression of the three enzymes of the L-Arg biosynthetic pathway: Ornithine Transcarbamylase (OTC), Argininosuccinate Synthase (ASS1) and Argininosuccinate Lyase (ASL) being required to convert ornithine to L-Arg. In a variety of tumor types, silencing of one or more of these enzymes disables endogenous L-Arg synthesis forcing a reliance on the extracellular pool of L-Arg for tumor survival and proliferation. This mechanism has been confirmed with Arginine Deiminase (ADI-PEG) and pegylated wild-type Arginase I (BCT-100). Aeglea Biotherapeutics Inc. has developed an alternative approach using a bioengineered human PEGylated Arginase I with enhanced pharmacological properties. Replacement of manganese, the natural metal co-factor in wild-type human Arginase I, with cobalt confers improved catalytic activity and serum stability. The resulting product candidate (AEB1102) displays highly favorable PK/PD properties, and is expected to be naturally tolerated by the human immune system as no protein modifications have been introduced. Experimental Procedures and Results: Non-clinical dose range finding and GLP toxicology studies were performed with AEB1102 in monkeys and mice. Bioanalytical assays detecting L-Arg and AEB1102 enzyme activity were used to monitor PD/PK in the dose range finding studies with these results subsequently being used to design the toxicology studies. Subsequent toxicology studies identified an NOAEL in both species at a dose that is predicted to translate to significant sustained reduction of L-Arg serum levels with weekly intravenous dosing. To determine tumor types most likely to respond to AEB1102 expression profiling of OTC, ASS1 and ASL was performed using in situ hybridization on multiple tumor histologies. Melanoma was identified as a tumor type likely to respond to AEB1102 owing to a significant reduction in ASS1 expression. Non-clinical in vivo studies using the A375 melanoma xenograft as well as melanoma PDx models confirm sensitivity to AEB1102, weekly dosing resulted in a significant delay in tumor growth and improved survival. Conclusion: Non-clinical activities required to support the IND submission of AEB1102 for solid tumors were successfully executed, enabling the Phase 1 study to be initiated in October 2015. Translational work profiling the expression of OTC, ASS1 and ASL has identified melanoma as a relevant tumor type to pursue in future clinical studies. Citation Format: Scott W. Rowlinson, Susan E. Alters, Giulia Agnello, Joseph Tyler, Ann Lowe, Mauri Okamoto-Kearney, Dale Johnson, Everett Stone, George Georgiou, David G. Lowe. Development of AEB1102, an engineered human arginase 1 for patients with solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1042.

Abstract 3964: Reducing systemic arginine with arginase (AEB1102) therapy does not suppress the immune response induced by anti-PD-1 and anti-PD-L1, and exerts an additive anti-tumor and synergistic survival benefit

Tumor dependence on specific amino acids for survival and proliferation is well recognized and has been exploited effectively with the use of asparaginases for the treatment of acute lymphoblastic leukemia. Sensitivity of tumors to L-Arginine (L-Arg) deprivation results from an impaired ability to make L-Arg as a result of decreased functional expression of one or more of the three enzymes of the L-Arg biosynthetic pathway: ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL). Native human arginase I is not a viable drug candidate due to low activity and low stability in serum. We have developed an alternative approach using a bioengineered human PEGylated arginase I (AEB1102) with enhanced pharmacological properties. We and others have successfully utilized arginase I to impart a direct tumor cell killing effect through L-Arg starvation in multiple tumor types e.g. AEB1102 single agent efficacy in melanoma, small cell lung cancer (SCLC) and sarcoma PDx models. However, the compatibility of AEB1102 with checkpoint inhibitors is unclear as arginase I has been shown to be both immune suppressive and immune neutral (PMID: 23717444), or immune promoting (PMID: 27043409). Because of these conflicting reports we decided to investigate the impact of systemic L-Arg removal on the anti-tumor efficacy of check point inhibitors. Murine syngeneic models (e.g. CT26, MC38 and LL2) were dosed with AEB1102 alone and in combination with anti-PD-L1 and anti-PD-1 monoclonal antibodies (mAbs). Depending on the model, in vivo treatment with AEB1102 monotherapy resulted in tumor growth inhibition (TGI) ranging from 52% to 72% compared to the untreated control group, whereas standard monotherapy using immunomodulatory mAbs that target PD-1 and PD-L1 resulted in TGI ranging from 12% to 60%. Of significance, combination therapy of AEB1102 with anti-PD-1 or anti-PD-L1 resulted in additive anti-tumor effect with TGI ranging from 60% to 86%. In the CT26 model, when AEB1102 was administered in combination with anti-PD-L1 for at least 6 weeks, a 33% frequency of complete tumor regression (non-palpable tumors) was observed, indicating that synergy occurs with combination therapy. Collectively these results demonstrate that disrupting the L-Arg physiological balance in the tumor microenvironment inhibits tumor growth and further sensitizes the tumor to immunotherapy when AEB1102 is combined with anti-PD-1 and anti-PD-L1. These data open the possibility of further improving outcomes in L-Arg dependent tumors through combination of AEB1102 with anti-PD-1 and anti-PD-L1 inhibitors. Citation Format: Giulia Agnello, Susan E. Alters, David G. Lowe, Scott W. Rowlinson. Reducing systemic arginine with arginase (AEB1102) therapy does not suppress the immune response induced by anti-PD-1 and anti-PD-L1, and exerts an additive anti-tumor and synergistic survival benefit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3964. doi:10.1158/1538-7445.AM2017-3964

Abstract 3073: Targeting chronic lymphocytic leukemia by interfering glutathione synthesis using a novel therapeutic enzyme cyst(e)inase (AEB3103)

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western countries. Despite recent advance in new therapeutic agents that have improved treatment outcomes, CLL remains incurable due in part to the inability to completely eradicate the leukemia cells in vivo. Previous studies showed that CLL cells have high intrinsic oxidative stress and are highly dependent on cellular antioxidant glutathione (GSH) to maintain redox balance and cell viability. One logical strategy to impact CLL cells would be to abrogate the glutathione protection of CLL cells in vivo. Recently we discovered that primary leukemia cells isolated from CLL patients were unable to effectively utilize cystine for GSH synthesis due to low expression of the cystine transporter Xc-, and that bone marrow stromal cells highly express Xc- and effectively take up cystine for conversion to cysteine (Zhang et al: Nature Cell Biology, 2012). These findings provide a biochemical basis to develop novel strategies to effectively target leukemia cells in the stromal microenvironment and improve in vivo therapeutic activity. In this study, we tested the depletion of extracellular cystine and cysteine using a novel therapeutic enzyme-cyst(e)inase (AEB3103), as a potential way to block GSH synthesis in CLL cells and abolish the stromal protection of the leukemia cells. Our study showed that AEB3103 was very effective in depleting GSH in CLL cells and caused massive CLL cell death even in the presence of stromal cells. Importantly, AEB3103 could also overcome drug resistance of CLL cells with p53 deficiency both in primary leukemia cells isolated from CLL patients with 17p deletion and mouse leukemic cells isolated from mouse model we recently reported (Liu et al: Leukemia, 2014). In addition, AEB3103 showed very low toxicity to normal cells. These promising in vitro data warrant further animal studies, which are currently on ongoing using the CLL mouse model with TCL1-Tg:p53-/- genotype. Our study suggests that AEB3103 and its combination with standard anti-CLL drugs may potentially be useful for clinical treatment of CLL, even for the more aggressive CLL subtypes with unfavorable cytogenetic alterations such as those with chromosome17p deletion and p53 mutations, and may improve in vivo therapeutic activity. Citation Format: Jinyun Liu, Li Feng, Everett M. Stone, Joseph Tyler, Scott W. Rowlinson, Michael J. Keating, Peng Huang. Targeting chronic lymphocytic leukemia by interfering glutathione synthesis using a novel therapeutic enzyme cyst(e)inase (AEB3103). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3073.