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Development of a Novel Multi-Isoform ALDH Inhibitor Effective As an Anti-Melanoma Agent

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Development of a Novel Multi-Isoform ALDH Inhibitor Effective As an Anti-Melanoma Agent Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Development of a novel multi-isoform ALDH inhibitor effective as an anti-melanoma agent Saketh S. Dinavahi1,8,9, Raghavendra Gowda1,7,8,9, Krishne Gowda1, Christopher G. Bazewicz3,8,9, Venkat R. Chirasani1, Madhu Babu Battu10, Arthur Berg5, Nikolay V. Dokholyan1,6, Shantu Amin1, and Gavin P. Robertson1,2,3,4,7,8,9. Departments of 1Pharmacology, 2Pathology, 3Dermatology, 4Surgery, 5Public Health Sciences, 6Biochemistry and Molecular Biology, 7Foreman Foundation for Melanoma Research, 8The Melanoma Center, 9The Melanoma Therapeutics Program, The Pennsylvania State University College of Medicine, Hershey, PA 17033; 10Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting and Diagnostics, Uppal, Hyderabad, India 500039. Running title: ALDH inhibition for melanoma. Corresponding author: Gavin P. Robertson, Department of Pharmacology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. Phone: (717) 531-8098; Fax: (717) 531-0480; E-mail: [email protected] Conflicts of interest: The authors declare no potential conflicts of interest. 1 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT The aldehyde dehydrogenases (ALDHs) are a major family of detoxifying enzymes that contribute to cancer progression and therapy resistance. ALDH overexpression is associated with a poor prognosis in many cancer types. The use of multi-ALDH isoform or isoform-specific ALDH inhibitors as anti- cancer agents is currently hindered by the lack of viable candidates. Most multi-ALDH isoform inhibitors lack bioavailability, are non-specific or toxic while most isoform-specific inhibitors are not effective as monotherapy due to the overlapping functions of ALDH family members. The present study details the development of a novel, potent, multi-isoform ALDH inhibitor, called KS100. The rationale for drug development was that inhibition of multiple ALDH isoforms might be more efficacious for cancer compared to isoform-specific inhibition. Enzymatic IC50s of KS100 were 207, 1,410 and 240 nM towards ALDH1A1, 2 and 3A1, respectively. Toxicity of KS100 was mitigated by development of a nanoliposomal formulation, called NanoKS100. NanoKS100 had a loading efficiency of ~69% and was stable long-term. NanoKS100 was 5-fold more selective for killing melanoma cells compared to normal human fibroblasts. NanoKS100 administered intravenously at a submaximal dose (3-fold lower) was effective at inhibiting xenografted melanoma tumor growth by ~65% without organ related toxicity. Mechanistically, inhibition by KS100 significantly reduced total cellular ALDH activity to increase reactive oxygen species generation, lipid peroxidation and accumulation of toxic aldehydes leading to apoptosis and autophagy. Collectively, these data suggest the successful preclinical development of a non-toxic, bioavailable, nanoliposomal formulation containing a novel multi-ALDH isoform inhibitor effective in the treatment of cancer. 2 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION. Malignant melanoma is an aggressive neoplasm accounting for the majority of skin cancer-related deaths (1). The outlook for metastatic disease remains poor, with current five-year survival rates of 20% (1). However, treatment strategies for malignant melanoma have vastly improved with the discovery of targeted therapies to BRAF and MEK along with the development of immune checkpoint inhibitors (2). Although current treatment strategies may kill the bulk of tumor cells, they often leave behind therapy-resistant cancer cells with a stem- cell-like phenotype, which serve as a reservoir for disease recurrence and metastasis (3). Cancer cells with stem cell characteristics comprise a small subset of undifferentiated cells that initiate tumor formation and generate multipotent progenitors (3). They promote tumor aggressiveness, repopulation after injury and metastasis, having intrinsic resistance to radiation, chemotherapy and targeted therapies (4). A major mechanism by which these cells develop resistance is through upregulation of the aldehyde dehydrogenases (ALDHs), which has impaired the response to preoperative chemotherapy and radiation in esophageal carcinoma (5), conventional chemotherapy, erlotinib and gefitinib in lung carcinoma (6), olaparib in breast carcinoma (7) and cyclophosphamide in a myriad of carcinomas (8,9). The 19 human ALDH isozymes are broadly defined as a superfamily of NAD(P)+-dependent enzymes that participate in aldehyde metabolism, catalyzing the oxidation of toxic aldehydes into carboxylic acids (10-13). ALDH activity within cells is generally a composite of the activities of multiple ALDH isoforms, which have overlapping substrate specificity (14,15). The ALDHs confer a survival advantage to metabolically active cancer cells, by oxidizing aldehydes that accumulate and cause oxidative damage, into less toxic, more soluble carboxylic acids (16,17). Accordingly, ALDH overexpression is linked to poorer survival in gastric, breast, lung, pancreatic and prostate carcinomas, as well as in head and neck squamous cell carcinomas (HNSCCs) (8,11,14,18,19). The ALDH1A1, 1A2, 1A3, 3A1 and 3A2 isozymes are particularly important in cancer progression and resistance to anti-cancer therapies (8,15,20,21). Current ALDH inhibitors can be categorized into multi-ALDH isoform inhibitors and isoform-specific inhibitors, which primarily inhibit one isoform (11). Limitations of multi-ALDH isoform inhibitors, such as N,N- diethylaminobenzaldehyde (DEAB), which targets ALDH1A1, 1A2, 1A3, 1B1, 2 and 5A1, 4-dimethylamino-4- methyl–pent-2-ynthioic acid-S-methylester (DIMATE), which targets ALDH1A1 and 3A1, and citral, which targets ALDH1A1, 1A3 and 2, are lack of bioavailability or toxicity (11). DIMATE has tumor inhibitory efficacy when injected intraperitoneally (i.p.) but will require further preclinical evaluation (22). More recently, the ALDH inhibitors (aldis) -1, -2, -3, -4, and -6 have been developed, which target ALDH1A1, 2 and 3A1, and show efficacy in killing cultured cancer cells, particularly as combinatorial therapy (23-25). However, these compounds have mainly been tested in vitro and thus require further validation in preclinical models (23-25). 3 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Isoform-specific inhibitors, such as Cpd 3 and CM037 (targeting ALDH1A1), CVT10216 (targeting ALDH2) and CB7 and CB29 (targeting ALDH3A1), have limited efficacy in killing cultured cancer cells, particularly when used as monotherapy, and have not been tested in animal cancer models (18,26,27). NCT-501, which targets ALDH1A1, has been shown to be effective in inhibiting HNSCC growth in animals via intra-tumoral injection, suggesting poor systemic bioavailability (28). Other more bioavailable ALDH1A1 specific inhibitors have been developed, such as the orally bioavailable compounds NCT-505 and NCT-506, and the i.p. available compounds 13g and 13h, but have not yet been evaluated in animals (29,30). Therefore, ALDH inhibitors are needed that inhibit the multiple functionally overlapping ALDH isoforms, with an acceptable pharmacological profile. The current study describes the design and development of a novel, potent, multi-isoform ALDH inhibitor, called KS100. KS100 was developed since ALDH1A1, 2 and 3A1 overexpression was observed in a cell line melanoma progression model and targeting these individual ALDH isoforms did not affect cultured cell growth. KS100 potently inhibited multiple ALDH isoforms with negligible toxicity when administered in a nanoliposomal form, called NanoKS100. NanoKS100 was bioavailable and inhibited melanoma tumor growth by ~65% at submaximal (3-fold lower) doses. Most importantly, KS100 significantly reduced total cellular ALDH activity compared to several ALDH inhibitors leading to enhanced reactive oxygen species (ROS) generation, lipid peroxidation and accumulation of toxic aldehydes causing increased apoptosis and autophagy. 4 Downloaded from mct.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 21, 2019; DOI: 10.1158/1535-7163.MCT-19-0360 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. MATERIALS AND METHODS. Cell lines, culture conditions, and chemicals: Normal human fibroblasts (FF2441) were provided by Dr. Craig Myers, Penn State College of Medicine, Hershey, PA. The human melanoma cell lines WM35, WM115, WM278, WM3211, 1205 Lu, and A375M and normal melanocytes (NHEM) were provided by Dr. Herlyn, Wistar Institute, Philadelphia,
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