bioRxiv preprint doi: https://doi.org/10.1101/2021.03.04.433676; this version posted March 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The M1 aminopeptidase NPEPPS is a 2 novel regulator of cisplatin sensitivity 3 4 Robert T. Jones1,15, Andrew Goodspeed1,3,15, Maryam C. Akbarzadeh2,4,16, Mathijs Scholtes2,16, 5 Hedvig Vekony1, Annie Jean1, Charlene B. Tilton1, Michael V. Orman1, Molishree Joshi1,5, 6 Teemu D. Laajala1,6, Mahmood Javaid7, Eric T. Clambey8, Ryan Layer7,9, Sarah Parker10, 7 Tokameh Mahmoudi2,11, Tahlita Zuiverloon2,*, Dan Theodorescu12,13,14,*, James C. Costello1,3,17,* 8 9 1Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, CO, 10 USA 11 2 Department of Urology, Erasmus MC Cancer Institute, Erasmus University Medical Center 12 Rotterdam, Rotterdam, The Netherlands 13 3University of Colorado Comprehensive Cancer Center, University of Colorado Anschutz 14 Medical Campus, Aurora, CO, USA 15 4Stem Cell and Regenerative Medicine Center of Excellence, Tehran University of Medical 16 Sciences, Tehran, Iran 17 5Functional Genomics Facility, University of Colorado Anschutz Medical Campus, Aurora, CO, 18 USA 19 6Department of Mathematics and Statistics, University of Turku, Turku, Finland. 20 7Computer Science Department, University of Colorado, Boulder 21 8Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, CO 22 9BioFrontiers Institute, University of Colorado, Boulder 23 10Smidt Heart Institute & Advanced Clinical Biosystems Research Institute, Cedars Sinai 24 Medical Center, Los Angeles, California 90048, United States 25 11Erasmus MC, Department of Biochemistry, Rotterdam, The Netherlands 26 12Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA 27 13Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, Los 28 Angeles, CA, USA 29 14Cedars-Sinai Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA 30 15Equal first authors 31 16These authors contributed equally 32 17Lead Contact 33 *Corresponding Authors 34 35 Corresponding Authors 36 Tahlita Zuiverloon, MD, PhD 37 Department of Urology 38 Erasmus MC Cancer Institute, Erasmus University Medical Center 39 Dr. Molewaterplein 40 40 3015GD, Rotterdam 41 The Netherlands 42 +31 6 26 41 90 87 43 [email protected] 44 45 Dan Theodorescu, MD, PhD 46 Department of Surgery and Pathology 47 Cedars-Sinai Medical Center 48 8700 Beverly Blvd. 49 OCC Mezz C2002 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.04.433676; this version posted March 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 3/23/21 10:12:00 PMLos Angeles, CA 90048 51 +1 (310) 423-8431 52 [email protected] 53 54 James C Costello, PhD 55 Department of Pharmacology 56 University of Colorado Anschutz Medical Campus 57 Mail Stop 8303 58 12801 E. 17th Ave., Rm L18-6114 59 Aurora, CO 80045 60 +1 (303) 724-8619 61 [email protected] 62 63 HIGHLIGHTS 64 65 • CRISPR screens with multi-omics identify NPEPPS as a driver of cisplatin resistance 66 • NPEPPS depletion in multiple bladder cancer models enhances cisplatin sensitivity 67 • LRRC8A and LRRC8D loss increase resistance to cisplatin in CRISPR screens 68 • Unique resource of functional and multi-omic data is provided to the community 69 70 KEY WORDS 71 72 NPEPPS; Volume Regulated Anion Channel; CRISPR Screen; Synthetic Lethality; multi-omics; 73 Bladder Cancer; DNA Repair; Cisplatin; Tosedostat 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.04.433676; this version posted March 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 74 ABSTRACT 75 76 Despite routine use of platinum-based chemotherapeutics across diverse cancer types, there 77 remains a need to improve efficacy and patient selection for treatment. A multi-omic 78 assessment of five human bladder cancer cell lines and their chemotherapy resistant 79 derivatives, coupled with whole-genome CRISPR screens were used to identify puromycin- 80 sensitive aminopeptidase, NPEPPS, as a novel functional driver of treatment resistance to 81 cisplatin. Depletion of NPEPPS resulted in enhanced cellular cisplatin import, sensitization of 82 resistant cancer cells to cisplatin in vitro and in vivo. Pharmacologic inhibition of NPEPPS with 83 tosedostat in cells and in chemoresistant, patient-derived tumor organoids improved response 84 to cisplatin. Depletion of LRRC8A and LRRC8D, two subunits of the volume regulated anion 85 channel (VRAC), a known importer of intracellular cisplatin, enhanced resistance to cisplatin. 86 Linking NPEPPS function to VRAC cisplatin import supports NPEPPS as a driver of cisplatin 87 resistance and by virtue of clinically available inhibitors, the potential for rapid clinical 88 translation. 89 90 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.04.433676; this version posted March 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 91 INTRODUCTION 92 93 Platinum-based chemotherapeutics have a long history (Dilruba and Kalayda, 2016; Rottenberg 94 et al., 2021) with successful applications in testicular, ovarian, bladder, head and neck, and lung 95 cancers. However, these drugs come with dose-dependent side effects that limit patient 96 eligibility. Additionally, chemoresistance mechanisms can arise, reducing the efficacy of these 97 drugs. While mechanisms of resistance have long been established, including DNA damage 98 repair and drug export (Galluzzi et al., 2012), other mechanisms, such as the import of platinum 99 drugs through volume regulated anion channels (VRACs) are more recently discovered and 100 present new opportunities for therapeutic development (Planells-Cases et al., 2015; Rottenberg 101 et al., 2021). Despite their limitations, platinum-based drugs remain the standard of care in 102 many cancer types and with a paucity of better treatment options for many patients, these drugs 103 will remain in use for the foreseeable future. Two avenues can be taken to improve patient 104 outcomes, which include discovery of more effective agents or development of strategies that 105 can improve efficacy of platinum-based regimens. The latter would have broad impact across a 106 range of cancer types. Here we take the latter approach and focus our efforts on bladder 107 cancer. 108 109 Bladder cancer (BCa) accounts for 430,000 new diagnoses and 170,000 deaths worldwide 110 annually (Bray et al., 2018). Cisplatin-based combination chemotherapy, in the form of 111 gemcitabine plus cisplatin (GemCis) or Methotrexate, Vinblastine, Adriamycin, and Cisplatin 112 (MVAC), remains the first-line, standard of care for metastatic BCa, providing a 5-10% cure rate. 113 However, up to 30% of patients are ineligible for cisplatin-based treatment (Galsky et al., 2018) 114 and are offered carboplatin-based combinations. Unfortunately carboplatin combination therapy 115 has been shown to be less effective in BCa (Patel et al., 2020). Alternatively, immune 116 checkpoint therapies (ICT) are being considered as a first-line therapy (Galsky et al., 2020); 117 however, ICT requires a PD-L1 diagnostic test, for which only ~25% patients meet eligibility 118 (Nadal and Bellmunt, 2019). On top of limited patient eligibility, the complete response rates for 119 ICT eligible patients is 20-30% (Balar et al., 2017), which limits the overall efficacy of ICT across 120 the population of patients with metastatic BCa. Cisplatin-based combination chemotherapy is 121 also standard of care in the neoadjuvant (NAC) setting for the management of localized muscle- 122 invasive bladder cancer (Grossman et al., 2003; Vale, 2005). However, NAC adoption has been 123 relatively slow due to the toxicity of the drugs, the number of patients that are cisplatin ineligible, 124 and the relatively small survival benefit of 5-15% over immediate cystectomy (Witjes et al., 125 2020). Importantly, in both the metastatic and NAC BCa settings, patient selection and 126 therapeutic efficacy of cisplatin-based chemotherapy are critical unresolved challenges (Patel et 127 al., 2020). 128 129 Recently, several large-scale efforts have performed whole genome loss-of-function screening 130 across hundreds of cancer cell lines using CRISPR- and shRNA-based libraries to define pan- 131 cancer and context-specific genetic dependencies (Cowley et al., 2014; McDonald et al., 2017; 132 Tsherniak et al., 2017; Behan et al., 2019). A limitation of these efforts in pharmacogenomics is 133 that cells were grown under basal growth conditions in the absence of treatment. Additionally, 134 those studies were performed in cell lines that had not necessarily acquired resistance to the 135 treatment. To better understand the functional drivers of therapeutic resistance, such screens 136 must be done in the presence and absence of the therapy of interest (Goodspeed et al., 2019; 137 Huang et al., 2020; Jost and Weissman, 2018; Olivieri et al., 2020), and in cells that have 138 acquired resistance to the treatment itself. Results from such synthetic lethal screens can be 139 used to prioritize gene candidates that can be targeted to overcome treatment resistance. 140 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.04.433676; this version posted March 24, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.