Targeted Inhibition of the E3 Ligase SCF Has Antitumor Activity in RB1

Published OnlineFirst April 7, 2020; DOI: 10.1158/0008-5472.CAN-19-2400

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deﬁcient Human and Mouse Small-Cell Lung Cancer Hongling Zhao1, Niloy J. Iqbal1, Vineeth Sukrithan2, Cari Nicholas1, Yingjiao Xue1, Cindy Yu1, Joseph Locker3, Juntao Zou1, Edward L. Schwartz2,4, and Liang Zhu1,5

ABSTRACT ◥ The RB1 tumor suppressor gene is mutated in highly aggressive knock-in of a mutant p27 that was unable to bind to Skp2. tumors including small-cell lung cancer (SCLC), where its loss, Building on the observed synthetic lethality between Rb1 and along with TP53, is required and sufficient for tumorigenesis. While Skp2, we found that small molecules that bind/inhibit Skp2 – RB1-mutant cells fail to arrest at G1 S in response to cell-cycle have in vivo antitumor activity in mouse tumors and human restriction point signals, this information has not led to effective patient-derived xenograft models of SCLC. Using genetic and strategies to treat RB1-deficient tumors, as it is challenging to pharmacologic approaches, antitumor activity was seen with develop targeted drugs for tumors that are driven by the loss of Skp2 loss or inhibition in established SCLC primary lung tumors, gene function. Our group previously identified Skp2, a substrate in liver metastases, and in chemotherapy-resistant tumors. Our recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early data highlight a downstream actionable target in RB1-deficient repression target of pRb whose knockout blocked tumorigenesis in cancers, for which there are currently no targeted therapies Rb1-deficient prostate and pituitary tumors. Here we used genetic available. mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice Significance: There are no effective therapies for SCLC. The (RP mice). Skp2 KO caused an increased accumulation of the identification of an actionable target downstream of RB1,inacti- Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, vated in SCLC and other advanced tumors, could have a broad which was confirmed as the mechanism of protection by using impact on its treatment.

Introduction been made in the treatment of SCLC. Except for recently reported modest increases in survival seen with the PD-L1 inhibitors nivolumab Small-cell lung cancer (SCLC) is characterized by aggressive growth, and atezolizumab, neither treatment options nor life expectancy have frequent metastases, and a 5-year survival of less than 5% (1, 2). While improved over the past three decades (1–4). it can be sensitive to first-line therapy of cisplatin and etoposide, most A unique feature of SCLC, not seen in any other solid tumor types, is patients rapidly relapse with chemotherapy-resistant tumors. Dozens the near uniform (>95%) biallelic inactivation of tumor suppressor of drugs have been tested clinically in SCLC, including more than 40 genes RB1 and TP53 to drive tumorigenesis (1, 2). This defining feature agents that have failed in phase III trials. The identification of driver of the disease has not led to a targeted therapy, however, because mutations and their corresponding targeted drugs have led to signif- genetically inactivated RB1 and TP53 cannot be reactivated, nor is it icant improvements in the treatment of non–small cell lung cancer feasible to clinically reintroduce the wild-type genes into all tumor cells (NSCLC) and other solid tumors; however, similar advances have not in vivo. The RB1 tumor suppressor gene governs multiple cellular functions, including proliferation, cell-cycle progression, and apoptosis, via a complex collection of molecular actions (5, 6). In lung cancers, 1Department of Developmental and Molecular Biology, Albert Einstein College loss of RB1 was associated with higher-grade tumors and an acceler- of Medicine, Bronx, New York. 2Department of Medicine (Oncology), Albert ation of metastatic competency (7). pRb regulates gene expression by 3 Einstein College of Medicine, Bronx, New York. Department of Pathology, binding to and suppressing the E2F1 transcription factor. The E2F University of Pittsburgh, Pittsburgh, Pennsylvania. 4Department of Molecular 5 family of transcription factors is important for cellular homeostasis, Pharmacology, Albert Einstein College of Medicine, Bronx, New York. Depart- – ment of Ophthalmology & Visual Sciences, Albert Einstein College of Medicine, and they are the major transcriptional regulators of cell-cycle Bronx, New York. dependent gene expression, particularly those genes required for the G –G to S-phase transition (8). Hence the loss of pRb leads to high Note: Supplementary data for this article are available at Cancer Research 1 0 Online (http://cancerres.aacrjournals.org/). E2F transcriptional activity, compromises the ability of cells to exit the cell cycle, and makes them highly susceptible to sustained proliferation Current address for J. Zou: Department of Internal Medicine, Jiangxi Provincial in the presence of activated oncogenes (6). Cancer Hospital, Nanchang, Jiangxi, China. While the ability of pRB to bind to E2Fs has been the focus of much Corresponding Authors: Edward L. Schwartz, Albert Einstein College of Med- research, protein interaction databases indicate that there are more icine, 1300 Morris Park Avenue, Block Building 614, Bronx, NY 10461. Phone: 718- than 300 proteins that might interact with pRB (6). For example, pRB 430-8864; Fax: 718-430-2044; E-mail: [email protected]; and fi Liang Zhu, [email protected] exerts signi cant cell-cycle control that is transcription-independent, due to its well-characterized regulation of protein stability by direct Cancer Res 2020;80:2355–67 effects on the ubiquitin-ligase proteasomal degradation pathway. This doi: 10.1158/0008-5472.CAN-19-2400 degradation pathway includes the SCF family of E3 ubiquitin ligases, 2020 American Association for Cancer Research. which play essential roles in the ubiquitination, degradation, and

AACRJournals.org | 2355

Zhao et al.

regulation of cellular protein turnover (9). SCF complexes consist of a Primary mouse SCLC lung cells (RP-Lung), primary mouse SCLC scaffold protein (Cul1), an adaptor protein (Skp1), an accessory liver metastatic cells (RP-LvMet), human SCLC cell lines, and protein (Cks1 aka Roc1), and an F-box component; the latter deter- human NSCLC cell lines mines the recognition specificity of the protein substrate(s) for Primary mouse RP-Lung cells and RP-LvMet cells were prepared ubiquitination. from 0.3 cm 0.3 cm pieces of lung or liver tumor tissues, which were Our group previously identified one of the SCF E3 ligases, minced and dissociated in collagenase A in 2 mL serum-free DMEM/ SCFSkp2/Cks1, as an early repression target of pRB, and found that F12 (Corning) for 30–60 minutes at 37C with gentle shaking. Then knockout of the Skp2 substrate recruiting subunit of SCFSkp2/Cks1 sample was diluted to 10 mL in serum-free DMEM/F12 and spun at complex effectively blocked tumorigenesis of pituitary melanotroph 200 g for 5 minutes. The cell pellet was resuspended in 1 mL trypsin þ/ tumors and thyroid C cells in Rb1 mice (10–13). Primary targets of (Gibco) and placed in a 37 C, 5% CO2 tissue culture incubator for 3 SCFSkp2/Cks1 (called Skp2 in this article) include the cyclin-dependent minutes. Sample was diluted into 20 mL serum-free DMEM/F12 and kinase inhibitor p27 (CDKN1b), a key cell-cycle regulator, which filtered through a 45-mm nylon cell strainer into a new tube. After delays progression from G1 phase into S-phase. The levels of p27 centrifuging (200 g) for 5 minutes, the pellet was resuspended in protein decline prior to cell entry into S-phase due to its Skp2- 5 mL of red cell lysis buffer and incubated for 5 minutes at room mediated ubiquitination and degradation. By binding to Skp2, pRb temperature. Cells were spun at 200 g for 5 minutes, and resus- prevents the binding, ubiquitination, and degradation of p27 by the pended with cell culture medium (DMEM/F12 containing 10% FBS, SCFSkp2/Cks1 complex. In cells that have lost functional pRB, therefore, 1% penicillin/streptomycin, and 1% glutamine). Cell genotypes were there is no restraint on Skp2-mediated p27 degradation, which leads to confirmed by PCR of Rb1, Trp53, Skp2 / and p27T187A/T187A. Human – the loss of key G1 S cell-cycle restriction point and promotes cell SCLC cell lines (H69, H146, H196, H446, and H720) and human proliferation. In fact, in some studies, cell-cycle control has been shown NSCLC cell lines (H520 and H460) were obtained from ATCC and to be better correlated with p27 levels than with changes in proteins were cultured in RPMI1640 (ATCC) containing 10% FBS, 1% pen- encoded by E2F-regulated genes (3). pRB also represses Skp2 mRNA icillin/streptomycin, and 1% glutamine. expression, and thus RB1 loss can lead to increased Skp2 levels that could further promote p27 degradation. Establishment of organoids In this report, we show that loss or inhibition of Skp2 can restore A primary SCLC tumor from an RP mouse was dissociated as some of the tumor suppressor actions of pRB in SCLC. The antitumor described above, and 2000 cells were embedded in growth factor– activities of small molecules that directly inhibit Skp2 activity or reduced Matrigel in 96 wells with DMEM/F12 medium containing prevent formation of an active SCFSkp2/Cks1 complex highlight poten- mitogens (EGF, FGF2, and FGF10), R-Spondin (Wnt/b-catenin sig- tial actionable targets in cancer cells that have lost RB1. We have naling agonist), Noggin and A83-01 (TGFb inhibitors), and Y-27632 validated the antitumor activity of the Skp2 inhibitors in vitro and (Rock inhibitor). The frequency of organoid forming units in the in vivo, using both human and mouse SCLC models. While previous dissociated suspension was approximately 0.1%, and this increased at studies have evaluated the therapeutic potential of Skp2 inhibitors, least 10 fold on the first passage (p1). Salient feature of the RP SCLC those studies focused on other tumor types or on the subset of tumors organoids were rapid growth of tightly packed small-sized cells. in which Skp2 was overexpressed (9, 14). In contrast, Skp2 inhibitors will theoretically have therapeutic effects on all SCLC, as RB1 loss and Growth inhibition assay presumed increased Skp2 activity occur in virtually all SCLC To determine the effects of the drugs on cell proliferation, cells were tumors (1, 2). plated overnight in 96 well plates at approximately 5 103 cells per well in 100 mL. They were treated with vehicle, compound C1 (Millipore Sigma), flavokawain A (FKA; LKT Labs), or pevonedi- Materials and Methods stat (DCTD/NCI and Millennium Pharmaceuticals) at various con- SCLC mouse models by Adeno-CMV-Cre intratracheal delivery. centrations. After 72 hours, cell numbers were determined with a Establishment of SCLC mouse models by intratracheal delivery of CellTiter-Glo 2.0 Luminescent Cell Viability Assay reagent (Promega). Adeno-CMV-Cre has been described previously (15). Rb1lox/lox; Plates were read for luminescence using a Fluostar Optima Lumin- lox/lox þ/þ lox/lox lox/lox / Trp53 ;Skp2 mice, Rb1 ;Trp53 ;Skp2 mice and ometer (B.M.G. Labtech) and the IC50 values were determined. Rb1lox/lox;Trp53lox/lox; p27T187A/T187A mice were used to establish SCLC To assess the effect of shRNA vectors on cell proliferation, 2 105 models. Mice about 8 weeks of age were anesthetized with ketamine/ cells were plated in 6-well plates in triplicate and treated with or xylazine and tumors were initiated by intratracheal delivery of 75 mLof without doxycycline. Cell numbers were determined by hemocytom- DMEM/F12 medium containing 2.5 107 PFU Adeno-CMV-Cre eter counting. (prepared by the Albert Einstein College of Medicine Gene Therapy Core) and 10 mmol/L CaCl2. Doxycycline-inducible Skp2 knockdown in primary RP-LvMet All animal procedures were reviewed and approved by the Einstein and H520 cells Institutional Animal Care and Use Committee (IACUC). Doxycycline-induced knockdown of Skp2 was done with a pTripZ lentiviral shRNA obtained from Dharmacon. The shRNA sequence In vivo CT for Skp2 knockdown in mouse primary RP-LvMet cells and human CT imaging was performed with an X-ray CT system (Latheta H520 cells were 50-GCAAGACTTCTGAACTGCTAT-3 and 50- LCT-200, Hitachi Aloka Medical). Mice were anesthetized with iso- TCAAATTTAGTGCGACTTA-30, respectively. An empty vector was flurane and imaged without any contrast reagent. Parameters used for used as a control. Lentiviral helper constructs psPAX2 and pMD2.G the CT scans were as follows: tube voltage: 50 kV; tube current: 0.5 mA; were gifts from Didier Trono (Addgene plasmid #12260 and #12259). axial field of view (FOV): 48 mm, with an inplane spatial resolution of Lentivirus stocks were generated and concentrated as described pre- 48 mm 48 mm and slice thickness of 100 mm. Qualitative analysis of viously (16). For lentivirus infection, cells were put in 1 mL virus- lung lesion areas was performed with LaTheta software (version 3.00). containing media (DMED/F12) with 8 mg/mL polybrene, and were

2356 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

then spun at 1,000 g for 30–60 minutes at 32C. Cells were using SmartSpec 3000 Spectrophotometer for equal loading by protein resuspended with fresh media and transferred into a culture plate content onto SDS-PAGE gels. Proteins were transferred to polyviny- fl and placed into a 37 C, 5% CO2 tissue culture incubator. Successful lidene di uoride (PVDF) membrane (IPVH00010, Millipore) and lentivirus transduction was ensured by puromycin (400-128p, probed with the following antibodies: Skp2 (sc-7164), p21 (sc-397), GeminiBio-Products) selection, followed by mRNA (qRT-PCR) and and a-tubulin (sc-8035), from Santa Cruz Biotechnology; cleaved protein (Western blot) measurements. Doxycycline (2 mg/mL; Sigma- caspase-3 (#9661) and p53 (#2524) from Cell Signaling Technology; Aldrich) was used to induce Skp2 knockdown in cultured cells. cullin-1 (ab75812) and p73 (ab40658) from Abcam; p27 (#610242, BD Primary cells from RP mice were never maintained in culture longer Biosciences), and pRb (554136, BD Pharmingen). than 10–14 days. For coimmunoprecipitation assays, cells were lysed in Nonidet P-40 buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L Reverse transcription and real-time quantitative PCR EDTA, 0.5% Nonidet P-40, and 10% glycerol] containing phosphatase RNA was extracted using RNeasy Mini Kit (74104, QIAGEN). and protease inhibitors. Precleared extracts were incubated with rabbit Oligo-dT and SuperScript II (Invitrogen) were used for the synthesis anti-Skp2 antibody. Immunocomplexes were recovered using protein of the first-strand cDNA at 42C for 60 minutes. SYBR green PCR A-agarose (P9269, Sigma), separated on SDS-polyacrylamide gels, and mixture (4309155, ABI) and the standard program of ABI 7500 Fast transferred to PVDF membrane. For Western blotting, the primary real-time PCR were used. GAPDH was used as internal control. The antibodies used were anti-Skp2 antibody and anti-p27 antibody. The qPCR primers sequences were: secondary antibody was horseradish peroxidase–labeled mouse anti- rabbit IgG (#3678S, Cell Signaling Technology). Proteins of interest mouse GAPDH-Forward: 50-AATGTGTCCGTCGTGGATCT-30; were detected using chemiluminescence (NEL104001EA, Perkin mouse GAPDH-Reverse: 50-GGTCCTCAGTGTAGCCCAAG-30, Elmer). mouse Skp2-Forward: 50-AGCAGCCGCTGGGTGAAAGC-30; mouse Skp2-Reverse: 50-ATCACTGAGTTCGACAGGTCCAT-30; IHC human GAPDH-Forward: 50-GGCCTCCAAGGAGTAAGACC-30; For IHC staining, tissues were fixed in 10% formalin, paraffin human GAPDH-Reverse: 50-AGGGGTCTACATGGCAACTG-30, embedded, and sectioned at 5-mm thickness. Slides were deparaffi- human Skp2-Forward: 50-TCCACGGCATACTGTCTCAG-30; nized, hydrated, and incubated in a steamer for 20 minutes in sodium human Skp2-Reverse: 50-GGGCAAATTCAGAGAATCCA-30. citrate buffer (Vector Labs) for antigen retrieval. Sections were first fi m The qPCR reactions were performed in a nal volume of 20 L. treated with 3% H2O2 for 10 minutes to quench endogenous perox- DD qPCR data were analyzed using Ct analysis method. All were done idase, washed with PBS for several times, blocked with 10% normal in triplicates and performed three separate times. goat serum for 1 hour, and then incubated in primary antibodies at 4C overnight. The following antibodies were used: Ki67 (ab16667, Establishment and treatment of orthotopic SCLC mouse Abcam,), cleaved caspase-3 (#9664S, Cell Signaling Technology), models, subcutaneous xenograft models, and PDX models synaptophysin (ab32127, Abcam), and chromogranin A (ab45179, Orthotopic SCLC models were established by intratracheal delivery Abcam). The sections were treated with SignalStain Boost IHC detec- of 1 106 primary mouse RP-LvMet cells (infected with doxycycline- tion reagent (Cell Signaling Technology) for 30 minutes. After three inducible shSkp2 lentivirus) into 6–8 weeks athymic nude mice (strain washes with PBS, the sections were incubated with SignalStain DAB code: 490, Charles River). Fifteen days later, mice were randomly substrate Kit (Cell Signaling Technology) was used to detect signals. divided into two groups for daily gavage treatment with 200 mLof IHC staining were counterstained with Harris Hematoxylin (Poly vehicle (control) or doxycycline (10 mg/mL). Primary mouse Scientific R&D Corp). Images were visualized with a Nikon Eclipse RP-LvMet cells (infected with doxycycline-inducible Skp2 knockdown Ti-U microscope and captured with Olympus DP71 camera and DP lentivirus) were used to establish subcutaneous (s.c.) tumors by Controller software (3.2.1.276), and saved with DP manager software injection of 1 106 cells into the flanks of athymic nude mice (3.1.1.208). The images were further processed by Adobe Photoshop. (strain code: 490, Charles River). When the tumor size was about Cleaved caspase-3 quantification was done using ImageJ software. 100–300 mm3, mice were divided into a control group and a group that was treated with doxycycline (5 mg/mL doxycycline in their Statistical analysis drinking water plus daily oral gavage of 1 mg doxycycline), FKA In the survival analysis, differences in Kaplan–Meier survival curves (600 mg/kg/day by oral gavage), or pevonedistat (50 or 90 mg/kg/day were analyzed by log-rank tests (Prism 6 Software). P < 0.05 was s.c.). Similarly, 1 106 H69 cells were injected subcutaneously into considered as statistically significant. nude mice that were then treated daily with vehicle or 40 mg/kg/day C1 (in DMSO) by intraperitoneal injection when the tumor size was about 100–300 mm3. For patient-derived xenograft (PDX) models, 1 1mm Results tumor pieces of tumors CTG-0199 or CTG-1252 (Champions Oncol- Loss of Skp2 is sufficient to prevent SCLC development in ogy) were inoculated sc into NCG mice (strain code: 572, Charles Rb1/Trp53–deficient mice River). When tumor volume reached about 100 mm3, mice were To study the role of Skp2 in SCLC, we used a mouse model randomized to vehicle or 600 mg/kg/day FKA treatment by gavage. (Rb1lox/lox;Trp53lox/lox) in which both alleles of the Rb1 and Trp53 Daily drug treatments continued for the duration of the experiments. genes are somatically deleted in the lung by intratracheal administra- The mice were monitored daily, tumor diameter was measured using tion of Adeno-Cre (17). These mice develop aggressive and metastatic calipers, and tumor volume was calculated as (length width2) 0.526. SCLC with histologic, pathologic, and neuroendocrine features that closely resemble advanced human SCLC (18). We generated cohorts of þ þ Western blots and coimmunoprecipitation assay Rb1lox/lox;Trp53lox/lox;Skp2 / (RP Skp2-WT SCLC) mice and litter- Cells or tumor masses (0.3 cm 0.3 cm) were lysed in RIPA buffer. mate Rb1lox/lox; Trp53lox/lox;Skp2 / (RP Skp2-KO) mice. At 8 weeks of Protein concentrations were determined by Bio-Rad protein Assay Kit age, Adeno-CMV-Cre was administered intratracheally to the mice to

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2357

Zhao et al.

delete Rb1 and Trp53, and initiate SCLC tumorigenesis (15). Male and NSCLC tumors metastasized to the liver in any of the groups, although female mice showed equal susceptibility to SCLC and our cohorts the other tumor types did. consist of equal numbers of both (17). At 4–6 months post Adeno-Cre inhalation, typical in situ lung lesions are found protruding into the Skp2 knockdown and inhibition inhibits the growth and mid-airway space, and these have small-cell morphology (scant cyto- metastasis of established SCLC tumors plasm), and stain positive for synaptophysin (Syp), a neuroendocrine While the lack of SCLC in RP Skp2-KO mice supports Skp20s SCLC marker, and Ki67 (Fig. 1A). Lungs were also examined by CT essential role in SCLC tumorigenesis, these experiments do not address scans (Supplementary Fig. S1A and S1B). At 8–10 months, multiple whether downregulation of Skp2 in established tumors would have a nodular tumors with SCLC morphology are detected throughout the beneficial effect, and could therefore serve as the basis for targeted drug lungs and the liver (Fig. 1B and F; Supplementary Figs. S1A and S2A; therapy. To answer this question, we avoided the long latency and Supplementary Table S1) as indicated by morphology, and synapto- confounding NSCLC and other tumor development of the RP trans- physin and chromogranin A staining. The incidence of SCLC in the genic mouse model, and used instead subcutaneous implantation of lung and livers of the RP mice with wild-type Skp2 was 91% and 74%, primary RP-LvMet cells (isolated from a SCLC metastatic liver respectively (Table 1). Both DNA (Supplementary Fig. S2B) and tumor from an RP mouse) to establish SCLC. During the brief period Western blot analyses (Supplementary Fig. S2C) confirmed the lung (< 2 weeks) that these cells were cultured, we infected them with an tumors were deficient for Rb1 and Trp53. The liver metastases in the inducible Skp2-shRNA lentivirus construct. In tissue culture, doxy- RP Skp2-WT mice stained positive for synaptophysin and chromo- cycline caused knockdown of Skp2, an increase in p27, a dramatic granin A, indicating they originated from the lung SCLC tumors decrease in cell proliferation, loss of viability, and the induction of cell (Fig. 1F). In contrast, RP Skp2-KO mice did not develop primary apoptosis (Fig. 2B and C). These cells were inoculated subcutaneously SCLC or SCLC liver metastasis, even at 24 months after Adeno-Cre in nude mice, and after allowing tumors to grow for 10 days to administration (Fig. 1C; Supplementary Fig. S1B and S2D; Table 1). approximately 100–300 mm3, we treated the mice with doxycycline The lung tumors in these mice were negative for synaptophysin and to knockdown Skp2 expression. The doxycycline-mediated knock- chromogranin A, but stained positive for the epithelial marker cyto- down of Skp2 had an antitumor effect in the subcutaneous xenograft keratin (Fig. 1G). mouse model, as illustrated by a significant decrease in tumor growth rates (Fig. 2D). Skp2 knockdown in these tumors was not complete p27 degradation mediates Skp2-dependent tumorigenesis in (Fig. 2E), which may account for the partial antitumor effect in vivo. Rb1/Trp53–deficient mice Mice were also inoculated orthotopically into the lung with the Because Skp2 targets a number of protein substrates for ubiquitina- inducible Skp2-shRNA RP-LvMet cells. The orthotopic tumors (Sup- tion, we determined which of these are key to SCLC development in the plementary Fig. S3A and S3B) showed SCLC pathology indistinguish- RP mice. As noted above, p27 is a well-established Skp2 substrate, and able from an autochthonous SCLC (Supplementary Fig. S3C and S3D). it must be phosphorylated on T187 for recognition by, and binding to, After allowing 15 days postinoculation for the tumors to become well the Skp2/Cks1 pocket. To test the role of p27 in Skp2-dependent SCLC, established, the mice were treated with doxycycline until they were we generated RP mice with a knock-in of a nonphosphorylatable sacrificed upon morbid appearance. Strikingly, while multiple large T187A-p27 (RP p27-AA; ref. 19). Because p27T187A cannot bind to and lesions were seen in the livers of 9 of 9 control mice in the absence of be ubiquitinated by Skp2, it will not be subject to degradation in RB1- doxycycline, only 2 of the 9 mice had liver metastases seen after mutant cells. As shown in Fig. 1D and G, Supplementary Fig. S2E Skp2 knockdown, and these liver metastases were smaller than and Table 1, there were no lung tumors with SCLC morphology or those seen in the control mice (Fig. 2F).Whetherthisisatrueanti- liver metastasis in these mice. We did find that mice in all three groups metastatic effect is uncertain, however, as it may reflect the lower developed NSCLC, probably due to loss of Trp53 in other lung cell primary tumor burden in the lungs of the Skp2 KD mice. These types (Fig. 1E and Table 1). findings are consistent with a recent report in which the reactivation Both RP Skp2-KO and RP p27-AA mice survived significantly of the RB pathway in RB1-deficient advanced lung adenocarcino- longer than RP Skp2-WT mice, with median survivals of 19 and mas caused their reprogramming toward a less aggressive state and 16 months for RP Skp2-KO and RP p27-AA mice, respectively, made them unlikely to metastasize (7). compared with 13 months for RP Skp2-WT mice (Fig. 2A). However, Our results show that SCLC is very sensitive to Skp2 knockdown. To this difference was not as dramatic as that seen in our studies of Rb1- determine whether this effect occurs in lung cancers that have not lost deficient pituitary melanotroph and thyroid C cell cancer (11). The RB1, we infected H520 NSCLC cells, which have wild-type RB1, with presence of NSCLC in the mice complicates the interpretation of the the same inducible Skp2-shRNA construct used in the SCLC cells. In survival data. In agreement with a previous report, the NSCLC tumors contrast to the SCLC cells (Fig. 2B), Skp2 knockdown by doxycycline had morphologies consistent with large cell, anaplastic cancer with did not affect the proliferation of the H520 cells (Fig. 2G) despite giant cells, as well as with adenocarcinomas, squamous carcinomas, causing a significant knockdown of Skp2 (Fig. 2H). Infection of the and tumors with mixed morphologies (Table 1; ref. 18). While the H520 cells with a constitutive shSkp2 similarly did not affect the incidence of NSCLC in the RP Skp2-WT mice (13.1%) was consistent proliferation of the cells. with the previous report, the Skp2-KO and p27AA mice had higher incidences of these lung tumors (75% and 53%, respectively). Other Small-molecule inhibitors of Skp2 inhibit SCLC proliferation malignancies, mainly with characteristics of leukemia and lymphomas, Having successfully employed genetic methods to define the role of were also observed;however, these were likely unrelated to the RP Skp2 in SCLC, we next used pharmacologic approaches to validate genotype (Table 1). There was a higher incidence of the other tumors Skp2 as a potential target for drug therapy. The crystal structure of the in the latter two groups as well (4.3% vs. 19% and 18%). It is possible SCF–Skp2–p27 complex has defined the interaction of Skp2 and Cks1 that the longer survival of these mice, due to the lack of SCLC tumors, to form a pocket to which p27T187 binds (14, 20–24). On the basis of allowed the eventual development of NSCLC and other tumor types, this information and using in silico modeling and virtual library and this may have been the cause of death in these mice. None of the screening, studies investigating inhibitors of Skp2 identified a small

2358 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

Figure 1. Induction of SCLC and NSCLC in RP (Rb1 and Trp53 double knockout) mice. A, In situ neuroendocrine airway lesion (arrow) in RP Skp2-WT lung 4.6 months post Adeno-Cre inhalation. Sections were stained with hematoxylin and eosin (H&E), synaptophysin (Syp), or Ki67, as indicated. B, Representative lung and liver from a RP Skp2-wild type (WT) mouse at 9.6 months post Adeno-Cre inhalation. Black arrows, SCLC lung tumors; box, NSCLC; arrowheads, liver metastasis. Lack of primary SCLC tumor or liver metastasis in a RP mouse in which Skp2 was also deleted (RP Skp2-KO), 14.3 months after Adeno-Cre (C), or in a RP p27-AA mouse at 11 months. NSCLC is indicated in the box (D). E, NSCLC (left) and SCLC (right) in the lung of a RP Skp2-WT mouse, showing positive staining for neuroendocrine markers (synaptophysin and chromogranin A) only in the SCLC. Scale bar, 50 mm (20 mm in inserts). F, IHC of liver metastasis from a RP Skp2-WT mouse showing positive staining for SCLC synaptophysin, chromogranin A, and Ki67. G, Immunostaining of lung tumors from RP Skp2-KO and RP p27-AA mice had positive staining for pan- cytokeratin (Pan-CK), an epithelial marker, but not for synaptophysin.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2359

Zhao et al.

Table 1. Lung and liver tumor incidence in RP Skp2-WT, RP Skp2-KO, and RP p27-AA mice from tissues obtained after Adeno-Cre administration.

Lung and liver tumors in each mouse genotype RP Skp2-WT RP Skp2-KO RP p27AA Lung Liver Lung Liver Lung Liver

Survival (months after Adeno-Cre)a 13.2 2.2 17.1 3.5c 15.0 3.7d Mice per group 23 23 16 11 28 16 SCLC: n (%) with tumors 21 (91%) 17 (74%) 0 0 0 0 Tumors per mouse (mean SD) 14 8.5 ND 0 0 0 0 NSCLC: n (%) with tumors Adenocarcinoma 0 0 3 (19%) 0 6 (21%) 0 Large cell anaplastic 2 (8.7%) 0 5 (31%) 0 4 (14%) 0 Squamous þ mixed squamous 0 0 1 (6%) 0 2 (7%) 0 Mixed histology 1 (4.3%) 0 3 (19%) 0 3 (11%) 0 Otherb 1 (4.3%) 4 (17%) 3 (19%) 3 (27%) 5 (18%) 8 (50%) No tumors 1 (4.3%) 2 (8.7%) 1 (6%) 8 (73%) 6 (21%) 8 (50%)

Note: Totals may exceed 100% due to multiple tumor types in some of the mice. Abbreviation: ND, not determined. aMean SD. bIncludes leukemias, lymphomas, and sarcomas. cSigniﬁcantly different from RP Skp2-WT, P < 0.0001. dSigniﬁcantly different from RP Skp2-WT, P ¼ 0.042.

molecule (called C1) that binds to the pocket, thereby preventing is not part of a SCFSkp2 complex, as the assembly of the complex binding of p27 to the Skp2/Cks1 pocket (Fig. 3A; ref. 14). As shown requires neddylated Cul 1, and therefore lacks E3 ligase activity, which in Fig. 3B, C1 inhibits proliferation of the human SCLC cell line H69 then leads to the large increase in p27. We confirmed this with and two primary cell lines we derived from a lung tumor and a liver coimmunoprecipitation assays showing that pevonedistat treatment metastasis in the RP mice (RP-Lung and RP-LvMet), but was 6- to 50- interrupted the interaction between Skp2 and p27 (Fig. 4D). The fold less active against H460 and H520 NSCLC cells. The average IC50 differences in Skp2 levels between pevonedistat and FKA treatment for C1 in a larger panel of SCLC cell lines was 2.78 mmol/L, which was may reflect the greater specificity for NAE of the former compound, as 2.6- to 12-fold lower than that for the NSCLC cells (Table 2). C1 FKA's activity is not restricted to the effects on NAE (27). increased p27 protein in human and murine SCLC cells but not in the Previous studies of panels of drugs on a large number of human two NSCLC cell lines (H460 and H520; Fig. 4A). Similarly, C1 induced SCLC cell lines found that the sensitivity of the cells to the drugs was apoptosis (cleaved caspase-3) in the SCLC cells but not in the NSCLC highly correlated with their response to etoposide, that is, cells that cells (Fig. 4A). While C1 has previously been tested against melanoma, were resistant to etoposide were generally resistant to all other breast, and prostate cancer cells, our study is the first to report the agents (28). We found this not to be the case for C1 or pevonedistat, fi activity of C1 against SCLC cells (14). however, as there was no signi cant correlation with their IC50s We next investigated a second approach to reduce Skp2 activity, compared with their IC50 for etoposide (Fig. 4E). The possibility that based on the requirement of the SCF complex to be neddylated for its the combination of C1 and etoposide could have synergistic antitumor assembly and stability (Fig. 3A; ref. 6). Neddylation is mediated by the activity was also investigated; however, no evidence for such combi- neddylation activating enzyme (NAE), which first helps release cullin natorial effects were observed. Although combining C1 with pevone- from its inhibitory protein CAND1, and then transfers NEDD8 (a distat in the SCLC cell lines did cause a significantly greater growth small ubiquitin-like protein) to cullin, which induces a structural inhibition than either agent alone, there was also no evidence that this rearrangement of cullin and the SCF complex, allowing the transfer interaction was synergistic (Fig. 4F). of ubiquitin from the E2 enzyme to the Skp2 protein substrate (24). We used two NAE inhibitors: flavokawain A (FKA), a chalcone derived Inhibitors of Skp2 have in vivo antitumor activity in mouse and from the kava plant that has been previously studied for its antitumor human SCLC effects in a number of cancer types, and pevonedistat (MLN4924), a Having demonstrated an inhibitory effect of the genetic knock- first-in-class inhibitor of NAE that has entered clinical trials (25–27). down of Skp2 on SCLC in vitro and in vivo,andthepharmacologic We found that both FKA and pevonedistat inhibited the prolifer- effect of Skp2 inhibition on SCLC in vitro, we next evaluated the ation of the H69, RP-Lung, and RP-LvMet SCLC cells (Table 2). In antitumor effects of the Skp2 inhibitors in vivo.Thethreecom- addition to inhibiting proliferation, pevonedistat caused cell death in pounds, C1, FKA, and pevonedistat, had significant antitumor preformed SCLC organoids, indicating its effects were cytotoxic and activity against SCLC subcutaneous xenografts in vivo.These not merely cytostatic (Supplementary Fig. S4). In contrast to C1, included the H69 human cell line (Fig. 5A), primary liver metastasis however, these agents showed less selectivity for SCLC, compared cells from the RP mouse (RP-LvMet; Fig. 5B and E), and two with the two NSCLC cell lines. As expected, both FKA and pevonedi- human SCLC PDXs (Fig. 5C and D). Both PDXs had mutant RB1 stat decreased the neddylation of Cul 1 (Fig. 4B) and increased p27 and and TP53, had characteristic small-cell morphology, and expressed p21 (Fig. 4C) in the SCLC cells. Interestingly, pevonedistat induced a SCLC neuroendocrine markers, as did the tumors derived from H69 large increase in Skp2 protein levels, in contrast to the previously and RP-LvMet (Fig. 5F; Supplementary Fig. S5). Inhibition of reported effect for FKA (Fig. 4C; ref. 27). It is presumed that this Skp2 tumor growth by the drugs was at least 50% in all cases, with

2360 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

Figure 2. Skp2 knockdown in established SCLC reduces tumor growth. A, Kaplan–Meier survival curves for the three mice groups described in Fig. 1. Survival was signiﬁcantly different between the groups by log-rank (Mantel–Cox) test. B and C, Primary tumor cells from liver metastasis from a RP mouse (RP-LvMet) were transduced with a lenti-shRNA lentivirus vector for inducible Skp2 knockdown (pTripZ-shSkp2) or a control virus. B, Cell growth in vitro with (&, ~, !) and without (*, ~) doxycycline (Dox) treatment for control (!, ~) and shSkp2 (*, &, !) vectors. C, Western blot showing treatment of the cells with 2 mg/mL doxycycline for 24 or 48 hours in vitro caused decreased expression of Skp2 and increased expression of p27 and cleaved caspase-3. D and E, H520 NSCLC cells were infected with the pTripZ-shSkp2 or a control virus. D, RP-LvMet cells with the pTripZ-shSkp2 vector were inoculated subcutaneously (SC) in nude mice and tumors allowed to grow for 10 days, at which time, tumors were 100-300 mm3. Half of the mice were treated with doxycycline (&; þDOX; 5 mg/mL doxycycline in their drinking water plus daily oral gavage of 1 mg doxycycline). Tumor sizes were measured every other day. Control mice (&; DOX) did not receive doxycycline. E, Skp2 mRNA levels in lung tumors from mice treated with and without doxycycline, measured by RT-PCR (n ¼ 5 mice). F, Hematoxylin and eosin–stained lung and liver from mice inoculated orthotopically in the lung with 106 Skp2-shRNA RP-LvMet SCLC cells. Treatment of the mice with DOX to knockdown Skp2 began on day 15 after inoculation, and the mice were sacriﬁced when moribund. G, Cell growth with (black bars) and without (gray bars) doxycycline. H, Skp2 mRNA levels by RT-PCR in H520 cells treated as in D.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2361

Zhao et al.

Figure 3. SCLC cells are sensitive to Skp2 inhibitors in vitro. A, Schematic showing the SCFSkp2 protein complex and its relationship to pRb, p27, and NEDD8. The sites of action of C1, FKA, and pevonedistat are shown. B, The indicated SCLC (green *, ~, !) and NSCLC (red *, ~) cells were plated in 96 well plates, and treated the next day with the indicated concentrations of C1. Cell numbers were quantiﬁed after 72 hours with a CellTiter-Glo assay.

increases in apoptosis and evidence of a cessation of growth with and paclitaxel chemotherapy, indicating that the Skp2 inhibitors prolonged drug treatment (Fig. 5A–J). RP-LvMet tumors from may be effective as a second-line therapy in chemo-resistant pevonedistat-treated mice had significant increases in Skp2, p27, tumors. There was no change in body weight in mice treated with and cleaved caspase-3 levels (Fig. 5G and H). It is noteworthy that C1 or pevonedistat (Fig. 5K). FKA was active in the CTG-0199 PDX (Fig. 5C), which was derived from a patient with SCLC who had previously received carboplatin Discussion Table 2. Calculation of drug IC50 from cells treated as in Fig. 3B. Despite having a high number of mutations and genomic alterations, second only among solid tumors to melanomas, SCLC has few IC (mmol/L) 50 obvious therapeutic targets for drug development, and no targeted Cell line C1 FKA Pevonedistat drugs have been shown to have clinical activity (29). While recent FDA H460 NSCLC 33 12 19 8.9 0.43 0.08 approval of PDL1 inhibitors represent the first new drugs approved for H520 NSCLC 7.3 2.1 18 11 >10 SCLC in over 20 years, their activity was modest, and the clinical RP-Lung SCLC 0.70 0.05 8.3 4.1 1.8 0.21 activity of checkpoint inhibitors seems to be less pronounced in SCLC RP-LvMet SCLC 0.63 0.16 12 2.7 0.18 0.08 than in other tumors with similarly high mutation burdens (1, 4). H69 SCLC 1.2 0.6 9.9 2.3 8.7 1.1 Recent genomic and proteomic profiling of SCLCs have identified H146 SCLC 5.3 0.8 ND 0.15 0.02 potential new targets, including genes involved in DNA repair, histone H720 SCLC 0.85 0.15 ND 0.015 0.005 H196 SCLC 8.0 2ND12 8 methylation, and the Notch pathway (1, 2). However, each of these may be suitable targets in only small subpopulations of patients with Note: Values are means SD of at least three experiments, each done in SCLC. In contrast, mutations and complex genomic translocations led triplicate. to biallelic inactivation of RB1 in 106 0f 108 human SCLC tumors that Abbreviation: ND, not determined. were examined, and the remaining two tumors had massive genomic

2362 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

Figure 4 Expression of p27 and Skp2 in treated cells. A, Western blot showing concentration-dependent increase in p27 in SCLC (RP-Lung, RP-LvMet, H69), but not in NSCLC (H460 H520) cells treated with C1 for 24 hours. B, FKA and pevonedistat reduced cullin1 neddylation in RP-Lung and H69 SCLC cells. C, Comparative effect of C1, FKA, and pevonedistat on Skp2, p27, p21, and cleaved caspase-3 protein expression in RP-Lung and H69 SCLC cells. D, Immunoprecipitation of Skp2 from control and

pevonedistat-treated cells showing loss of coimmunoprecipitated p27 in drug-treated cells. E, Lack of correlation between the IC50s for etoposide with those for C1 (*) and pevonedistat (*) in seven SCLC cell lines. F, Effect of the combination of C1 and pevonedistat on cell proliferation in SCLC cells. Data are the means SD from

ﬁve cell lines (H69, H146, H196, H446, H720). Each cell line was treated for 72 hours with C1 or pevonedistat at their respective IC50s, with the drugs used alone or in combination. , P < 0.05, signiﬁcant difference between the drugs used in combination compared with their use individually.

rearrangements (chromothripis) accompanied by the loss of pRB The activity of pRb is controlled by cyclin-dependent kinases, expression (30). The importance of the universal inactivation of RB1 and high levels of active pRb are found in G1 phase and quiescent in SCLC was conﬁrmed in genetically engineered mouse models, in cells (31). While the role of pRb in regulating cell-cycle arrest at the – which the somatic biallelic deletion of Rb1 and Trp53 was both G1 S restriction point has been well-established, questions have required and sufﬁcient for the development of aggressive and meta- arisen as to whether this regulation is solely due to pRb's effect on static neuroendocrine lung cancers (18). E2F transcription factors and the transcription of cell-cycle

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2363

Zhao et al.

Figure 5. SCLC tumors are sensitive to Skp2 inhibitors in vivo. A–E, Tumors were initiated by the subcutaneous inoculation of human H69 SCLC cells (A), murine RP-LvMet SCLC cells (B and E), and 1-2 mm fragments of the human SCLC PDXs CTG-0199 (C) and CTG-1252 (D). When tumors reached 100 mm3 in size, mice were treated with C1 (40 mg/kg/day i.p.; A), FKA (600 mg/kg/day by gavage; B–D), or pevonedistat (50 or 90 mg/kg/day s.c.; E). Tumor size was measured on the indicated days; data are means SEM of at least 5 mice per group. All drug treatments were signiﬁcantly different from controls (P < 0.0001). F, Both PDXs stained positive for neuroendocrine markers synaptophyosin (Syp) and chromogranin A (ChromA), and had a large population of actively proliferating Ki67-positive cells. G, Tumors isolated from drug-treated mice had increased apoptosis after staining for cleaved caspase-3 IHC. H and I, Western blot and quantitation of Skp2, p27, and cleaved caspase-3 in RP-LvMet tumor extracts from control and pevonedistat-treated mice. Each lane in H is an individual mouse tumor. J, Quantitation of cleaved caspase-3 (percent positive staining cells) in RP-LvMet and PDX tumors isolated from control or drug-treated mice using ImageJ software. K, Mouse body weight during daily treatment with C1 or pevonedistat, as indicated. Means SD of 5-6 mice per group.

2364 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

genes (6). Pinpointing the specificfunctionofpRb,whoselossis compared with NSCLC cells, suggests that C1 has a degree of critical for driving tumorigenesis in RB1-deficient cells, is an selectivity and could serve as a prototype for additional medicinal essential first step for the identification of a downstream pathway chemistry optimization. Our evidence for in vivo antitumor activity for drug targeting. Our previous reports that the loss of Skp2 was in a mouse SCLC model further support this conclusion. Further sufficient to prevent tumorigenesis in Rb1 knockout mice supported studies are needed to determine if the noted selectivity is due to the acriticalroleforatranscription-independent action of pRb, that is, loss of Rb1. the regulation of protein stability by the ubiquitin–proteasome While directly targeting the p27–Skp2–Cks1 interface has the system (12, 19). advantage of high specificity for the intended target, as discussed pRb directly binds to the N terminus of Skp2 in the SCF complex, above, to date only compounds of modest potency have been iden- which causes the repression of Skp2-E3 ligase activity. pRb binding tified. We therefore looked for pathways responsible for the formation blocks the binding of p27 to Skp2-SCF, prevents the ubiquitination and and/or stability of the Skp2 complex for which there were known degradation of p27, and maintains cell-cycle arrest. Hence the loss of potent inhibitors. Blocking the neddylation of the complex on cullin 1 RB1 results in unregulated activation of Skp2, unregulated loss of p27, was one such approach that we reasoned could result in a decrease in and reduced cell-cycle control. The importance of this action of pRb active Skp2 levels. FKA is a NAE inhibitor that works in RB1-deficient was shown using a pRb mutant that could not bind to E2F but retained prostate cancer cell lines by binding to the ATP-binding site of the full activity in inhibiting the Skp2–p27 interaction and inducing cell- APPBP–UBA3–NEDD8–ATP quaternary complex and inhibiting its cycle arrest (3). In the absence of RB1, Skp2 can act as an oncogene, a formation (37). Because FKA is a relatively nonspecific and low conclusion supported by findings that Skp2 gene is amplified in 44%, potency inhibitor of NAE and Cul1 neddylation, its clinical utility is and overexpressed in 75% of human RB1-mutant SCLC (32, 33). uncertain. Although loss of pRb leads to E2F1 activation, we previously found that We also tested pevonedistat, a highly potent (1,000-fold higher cyclin A, a target of E2F1, restrained the activity of E2F1. When Skp2 is compared with FKA) and specific neddylation inhibitor. In human codeleted with Rb1, accumulated p27 sequesters cyclin A away from cancer cell lines, pevonedistat inhibits modification of cullin proteins the E2F1 promotor, which not only potently activates E2F1 target (including in SCF-Skp2) by NEDD8, resulting in increased levels of genes but also converts an oncogenic E2F1 into a proapoptotic E2F1 CRL (cullin-ring-ligase) ubiquitination substrates, but not substrates via activating p73 expression (10, 34, 35). Increased accumulation of ubiquitinated by other non-cullin RING E3 ligases (Fig. 3A). In the Skp2 degradation target p27 transforms the oncogenic pRb contrast to proteasome inhibitors with broad activity (e.g., bortezo- repression target E2F1 to an apoptotic E2F1, revealing synthetic mib), it suppresses only a small subset of intracellular protein turnover. lethality between Rb1 and Skp2, which remained unabated when Thus, through modulation of NAE, the NEDD8 pathway regulates Trp53 was additionally deleted (10, 13). abundance of Skp2 and its substrates. Other mechanisms of action are Understanding the nature of the interaction between Skp2, pRb, also possible, for example, the neddylation of E2F1 controls its target and p27 is valuable not only for defining the basis for the tumor specificity such that its deneddylation switches it from stimulating suppression actions of RB1, but also to aid in the identification of proliferation to promoting apoptosis (38). Of relevance to SCLC, p53- potential target sites for drug design. The assembly of four compo- deficient cancer cells may be more sensitive to pevonedistat than those nents forms a functional SCFSkp2: Skp2, its adaptor protein (Skp1), with unmodified p53 (39). its accessory protein (Cks1), and its substrate (p27). While SCFSkp2 The importance of the neddylation pathway in SCLC is supported lacks the traditional catalytic sites of other enzymes that allows for by studies that found dysregulation of the levels of CAND1 (decreased direct inhibitor design, it does have biochemically distinct potential expression), Cul1 (increased), neddylated Cul1 (increased), and Skp2 drug–binding pockets that could serve as targets for small-molecule (increased) in human SCLC specimens (24–26). Increasing the levels design (9, 36). The design of drugs targeting these pockets requires of SCF-Skp2 in SCLC cells could amplify the effect of loss of RB1 on detailed structural analysis of the relevant protein–protein inter- p27 degradation and cell-cycle regulation. Consequently, by reducing actions, and this knowledge is incomplete as there are currently no the levels of Skp2, a neddylation inhibitor could indirectly cause p27 X-ray cocrystal structures of small-molecule inhibitors bound to accumulation, and when used in combination with a direct Skp2 SCFSkp2. inhibitor, lead an additive or better inhibitory effect on SCLC. How- Data that are available suggest our understanding of the complex, ever, we found no evidence that the combination had complementary based on the crystal structure of the Skp1/Skp2/Cks1 subunits effects on the inhibition of cell proliferation compared with the drugs combined with a peptide representing the binding region of p27, used alone. may not fully reflect the in vivo structure (36). Nevertheless, a Dependency Map analysis to identify genetic dependencies found suitable pocket was identified at the p27–Skp2–Cks1 interface, and a the Skp2 is a pan-essential gene (505/625 of CRISPR Common molecule(C1)thatbindstothispocketandinhibitsSkp2ligase Essential Genes) (depmap.org/portal/gene). While this analysis also activity has been identified using virtual library screening (14, 36). found a highly significant (P ¼ 2.7 10 5) enrichment of Skp2 While other potential binding pockets and inhibitory small mole- sensitivity in SCLC, it also raises the possibility that its inhibition could cules have been reported, C1 is one of only two molecules that have deleterious effects on normal cells. Studies of mice with a targeted docked to its expected target site using ICM-Dock, a docking deletion of Skp2 (Skp2 / ) mice suggest that any untoward effects algorithm shown to have the best accuracy when compared with would be manageable, particularly in the context of a relatively short other algorithms (36). Our finding that C1 significantly increases inhibitor treatment. The Skp2 / mice showed no evidence of illness or p27 levels in SCLC cells is consistent with physical data showing gross anatomic abnormalities up to 10 months of age, and both male that C1 binds specifically to the Skp2–Cks1 protein complex, that and female were fertile (40). The body weights of the Skp2 / mice this binding sterically clashes with the interaction of p27 with were reduced compared with their littermate controls, but this seems to Skp2-Cks1,andthatitcausesaconcentration-dependent inhibition reflect changes occurring during embryogenesis that would not be of the ligase activity of SCFSkp2 in a cell-free system (14). Our relevant in the treatment of adults with a Skp2 inhibitor. Cellular finding of a 2.6- to 12-fold greater sensitivity to C1 of SCLC cells, changes noted include centrosome overduplication and accumulation

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2365

Zhao et al.

of p27 and cyclin E, and the consequences of these on normal cells Disclosure of Potential Conflicts of Interest would have to be monitored during drug treatment. In our studies with No potential conflicts of interest were disclosed. the Skp2 inhibitor C1, we did not find any changes in body weight in treated mice (Fig. 5K). Authors’ Contributions Our previous work documented the importance of Skp2 in pituitary Conception and design: H. Zhao, N.J. Iqbal, E.L. Schwartz, L. Zhu and prostate cancers driven by the loss of Rb1, and we have now Development of methodology: J. Locker extended these observations to SCLC using molecular, genetic, and Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Zhao, N.J. Iqbal, V. Sukrithan, C. Nicholas, Y. Xue, J. Locker translationally relevant pharmacologic approaches to demonstrate Analysis and interpretation of data (e.g., statistical analysis, biostatistics, that Skp2 can act as a therapeutic target for the treatment of advanced computational analysis): H. Zhao, N.J. Iqbal, C. Nicholas, J. Locker, J. Zou, SCLC. We have identified and validated a downstream actionable E.L. Schwartz target of pRb in SCLC, and show that small molecules inhibiting this Writing, review, and/or revision of the manuscript: H. Zhao, N.J. Iqbal, C. Nicholas, newly defined signaling pathway slows tumor growth in human and J. Locker, E.L. Schwartz, L. Zhu mouse SCLC. While our data show a consistent synthetic lethal Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Nicholas interaction between RB1 and Skp2, whether Skp2 inhibition would Study supervision: E.L. Schwartz, L. Zhu be also effective in tumors with wild-type RB1 remains an open Other (assisted with production of lentivirus, for skp2 knockdown): C. Yu question. While our data from the H520 cell line are consistent with a selective effect based on RB1, Skp2 knockdown would need to be Acknowledgments evaluated in additional cell lines before a definitive answer can be We thank Angela Davies and Champions Oncology for providing the SCLC obtained. In addition to SCLC, RB1 is frequently mutated in other solid PDXs and the NCI Division of Cancer Treatment and Diagnosis and Millennium tumors, including advanced gastroenteropancreatic neuroendocrine Pharmaceuticals for providing pevonedistat. H. Zhao, N. Iqbal, V. Sukrtithan, C. Nicholas, Y. Xue, C. Yu, E.L. Schwartz and L. Zhu received support from NIH carcinomas, osteosarcomas, and metastatic breast cancers, and this is RO1CA230032. This work was supported in part by an NIH Cancer Center usually associated with poor prognosis and resistance to most che- Support grant to the Albert Einstein Cancer Center (CA013330). motherapy drugs (41–43). Interestingly, recent proteogenomic analysis of the paradoxical amplification of RB1 seen in some human colon The costs of publication of this article were defrayed in part by the payment of page cancers was found to be associated with pRb hyperphosphorylation charges. This article must therefore be hereby marked advertisement in accordance and the consequent inactivation of pRb, suggesting that select colo- with 18 U.S.C. Section 1734 solely to indicate this fact. rectal cancers could also be candidates for anti-Skp2 therapy (44). Thus, the use of Skp2 inhibitors could have an impact on a broad range Received August 2, 2019; revised January 20, 2020; accepted March 30, 2020; of advanced and intractable cancers. published first April 7, 2020.

References 1. Sabari JK, Lok BH, Laird JH, Poirier JT, Rudin CM. Unravelling the biology of 14. Wu L, Grigoryan AV, Li Y, Hao B, Pagano M, Cardozo TJ. Specific small SCLC: implications for therapy. Nat Rev Clin Oncol 2017;14:549–61. molecule inhibitors of Skp2-mediated p27 degradation. Chem Biol 2012;19: 2. Gazdar AF, Bunn PA, Minna JD. Small-cell lung cancer: what we know, what we 1515–24. need to know and the path forward. Nat Rev Cancer 2017;17:725–37. 15. DuPage M, Dooley AL, Jacks T. Conditional mouse lung cancer models using 3. Ji P, Jiang H, Rekhtman K, Bloom J, Ichetovkin M, Pagano M, et al. An Rb-Skp2- adenoviral or lentiviral delivery of Cre recombinase. Nat Protoc 2009;4:1064–72. p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a 16. Sun D, Melegari M, Sridhar S, Rogler CE, Zhu L. Multi-miRNA hairpin method partial-penetrance Rb mutant. Mol Cell 2004;16:47–58. that improves gene knockdown efficiency and provides linked multi-gene 4. Hiatt JB, MacPherson D. Delivering a STINGing blow to small cell lung cancer knockdown. BioTechniques 2006;41:59–63. via synergistic inhibition of DNA-damage response and immune-checkpoint 17. Meuwissen R, Linn SC, Linnoila RI, Zevenhoven J, Mooi WJ, Berns A. Induction pathways. Cancer Discov 2019;9:584–6. of small cell lung cancer by somatic inactivation of both Trp53 and Rb1 in a 5. Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the conditional mouse model. Cancer Cell 2003;4:181–9. retinoblastoma gene. Nat Rev Cancer 2008;8:671–82. 18. Gazdar AF, Savage TK, Johnson JE, Berns A, Sage J, Linnoila RI, et al. The 6. Dyson NJ. RB1: a prototype tumor suppressor and an enigma. Genes Dev 2016; comparative pathology of genetically engineered mouse models for neuroen- 30:1492–502. docrine carcinomas of the lung. J Thorac Oncol 2015;10:553–64. 7. Walter DM, Yates TJ, Ruiz-Torres M, Kim-Kiselak C, Gudiel AA, Deshpande C, 19. Zhao H, Lu Z, Bauzon F, Fu H, Cui J, Locker J, et al. p27T187A knockin identifies et al. RB constrains lineage fidelity and multiple stages of tumour progression Skp2/Cks1 pocket inhibitors for advanced prostate cancer. Oncogene 2017;36: and metastasis. Nature 2019;569:423–7. 60–70. 8. Kent LN, Leone G. The broken cycle: E2F dysfunction in cancer. Nat Rev Cancer 20. Chan CH, Morrow JK, Li CF, Gao Y, Jin G, Moten A, et al. Pharmacological 2019;19:326–38. inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and 9. Skaar JR, Pagan JK, Pagano M. SCF ubiquitin ligase-targeted therapies. Nat Rev cancer progression. Cell 2013;154:556–68. Drug Discov 2014;13:889–903. 21. Hao B, Zheng N, Schulman BA, Wu G, Miller JJ, Pagano M, et al. Structural basis 10. Lu Z, Bauzon F, Fu H, Cui J, Zhao H, Nakayama K, et al. Skp2 suppresses of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin apoptosis in Rb1-deficient tumours by limiting E2F1 activity. Nat Commun ligase. Mol Cell 2005;20:9–19. 2014;5:3463. 22. Kitagawa K, Kitagawa M. The SCF-type E3 ubiquitin ligases as cancer targets. 11. Wang H, Bauzon F, Ji P, Xu X, Sun D, Locker J, et al. Skp2 is required for survival Curr Cancer Drug Targets 2016;16:119–29. of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1 23. Lee Y, Lim HS. Skp2 Inhibitors: Novel Anticancer Strategies. Curr Med Chem mice. Nat Genet 2010;42:83–8. 2016;23:2363–79. 12. Zhao H, Bauzon F, Fu H, Lu Z, Cui J, Nakayama K, et al. Skp2 deletion unmasks a 24. Singh R, Sran A, Carroll DC, Huang J, Tsvetkov L, Zhou X, et al. Developing p27 safeguard that blocks tumorigenesis in the absence of pRb and p53 tumor structure-activity relationships from an HTS hit for inhibition of the Cks1-Skp2 suppressors. Cancer Cell 2013;24:645–59. protein-protein interaction. Bioorg Med Chem Lett 2015;25:5199–202. 13. Zhao H, Wang H, Bauzon F, Lu Z, Fu H, Cui J, et al. Deletions of retinoblastoma 1 25. Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An (Rb1) and its repressing target S phase kinase-associated protein 2 (Skp2) are inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature synthetic lethal in mouse embryogenesis. J Biol Chem 2016;291:10201–9. 2009;458:732–6.

2366 Cancer Res; 80(11) June 1, 2020 CANCER RESEARCH

Skp2 E3 Ligase Is an Actionable Target in RB1-Deﬁcient SCLC

26. Swords RT, Coutre S, Maris MB, Zeidner JF, Foran JM, Cruz J, et al. Pevonedistat, 36. Lough L, Sherman D, Ni E, Young LM, Hao B, Cardozo T. Chemical probes of a first-in-class NEDD8-activating enzyme inhibitor, combined with azacitidine Skp2-mediated p27 ubiquitylation and degradation. Medchemcomm 2018;9: in patients with AML. Blood 2018;131:1415–24. 1093–104. 27. Zi X, Simoneau AR. Flavokawain A, a novel chalcone from kava extract, induces 37. Li X, Yokoyama NN, Zhang S, Ding L, Liu HM, Lilly MB, et al. Flavokawain A apoptosis in bladder cancer cells by involvement of Bax protein-dependent and induces deNEDDylation and Skp2 degradation leading to inhibition of tumor- mitochondria-dependent apoptotic pathway and suppresses tumor growth in igenesis and cancer progression in the TRAMP transgenic mouse model. mice. Cancer Res 2005;65:3479–86. Oncotarget 2015;6:41809–24. 28. Polley E, Kunkel M, Evans D, Silvers T, Delosh R, Laudeman J, et al. Small cell 38. Aoki I, Higuchi M, Gotoh Y. NEDDylation controls the target specificity of E2F1 lung cancer screen of oncology drugs, investigational agents, and gene and and apoptosis induction. Oncogene 2013;32:3954–64. microRNA expression. J Natl Cancer Inst 2016;108. DOI: 10.1093/jnci/djw122. 39. Lin JJ, Milhollen MA, Smith PG, Narayanan U, Dutta A. NEDD8-targeting drug 29. Pietanza MC, Byers LA, Minna JD, Rudin CM. Small cell lung cancer: will recent MLN4924 elicits DNA rereplication by stabilizing Cdt1 in S phase, triggering progress lead to improved outcomes? Clin Cancer Res 2015;21:2244–55. checkpoint activation, apoptosis, and senescence in cancer cells. Cancer Res 30. George J, Lim JS, Jang SJ, Cun Y, Ozretic L, Kong G, et al. Comprehensive 2010;70:10310–20. genomic profiles of small cell lung cancer. Nature 2015;524:47–53. 40. Nakayama K, Nagahama H, Minamishima YA, Matsumoto M, Nakamichi I, 31. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81: Kitagawa K, et al. Targeted disruption of Skp2 results in accumulation of cyclin E 323–30. and p27(Kip1), polyploidy and centrosome overduplication. EMBO J 2000;19: 32. Dobashi Y, Tsubochi H, Minegishi K, Kitagawa M, Otani S, Ooi A. Regulation of 2069–81. p27 by ubiquitin ligases and its pathological significance in human lung 41. Pizzi S, Azzoni C, Bassi D, Bottarelli L, Milione M, Bordi C. Genetic alterations in carcinomas. Hum Pathol 2017;66:67–78. poorly differentiated endocrine carcinomas of the gastrointestinal tract. Cancer 33. Yokoi S, Yasui K, Saito-Ohara F, Koshikawa K, Iizasa T, Fujisawa T, et al. A novel 2003;98:1273–82. target gene, SKP2, within the 5p13 amplicon that is frequently detected in small 42. Bertucci F, Ng CKY, Patsouris A, Droin N, Piscuoglio S, Carbuccia N, et al. cell lung cancers. Am J Pathol 2002;161:207–16. Genomic characterization of metastatic breast cancers. Nature 2019;569:560–4. 34. Yamasaki L, Bronson R, Williams BO, Dyson NJ, Harlow E, Jacks T. Loss of E2F- 43. Ren W, Gu G. Prognostic implications of RB1 tumour suppressor gene altera- 1 reduces tumorigenesis and extends the lifespan of Rb1()mice. Nat Genet tions in the clinical outcome of human osteosarcoma: a meta-analysis. Eur J 1998;18:360–4. Cancer Care 2017;26. 35. Ziebold U, Lee EY, Bronson RT, Lees JA. E2F3 loss has opposing effects on 44. Vasaikar S, Huang C, Wang X, Petyuk VA, Savage SR, Wen B, et al. Proteo- different pRB-deficient tumors, resulting in suppression of pituitary tumors but genomic analysis of human colon cancer reveals new therapeutic opportunities. metastasis of medullary thyroid carcinomas. Mol Cell Biol 2003;23:6542–52. Cell 2019;177:1035–49.e19. DOI: 10.1016/j.cell.2019.03.030.

AACRJournals.org Cancer Res; 80(11) June 1, 2020 2367

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deficient Human and Mouse Small-Cell Lung Cancer

Hongling Zhao, Niloy J. Iqbal, Vineeth Sukrithan, et al.

Cancer Res 2020;80:2355-2367. Published OnlineFirst April 7, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-19-2400

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2020/04/07/0008-5472.CAN-19-2400.DC1

Cited articles This article cites 42 articles, 9 of which you can access for free at: http://cancerres.aacrjournals.org/content/80/11/2355.full#ref-list-1

Citing articles This article has been cited by 1 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/80/11/2355.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/80/11/2355. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.