Abemaciclib, a Selective CDK4/6 Inhibitor, Enhances The

Published OnlineFirst May 1, 2018; DOI: 10.1158/1078-0432.CCR-17-3575

Cancer Therapy: Preclinical Clinical Cancer Research Abemaciclib, a Selective CDK4/6 Inhibitor, Enhances the Radiosensitivity of Non–Small Cell Lung Cancer In Vitro and In Vivo Sarwat Naz, Anastasia Sowers, Rajani Choudhuri, Maria Wissler, Janet Gamson, Askale Mathias, John A. Cook, and James B. Mitchell

Abstract

Purpose: To characterize the ionizing radiation (IR) enhancing sensitivity was lost in cell lines deficient for functional p53 and RB effects and underlying mechanisms of the CDK4/6 inhibitor protein. After IR, abemaciclib treatment inhibited DNA damage abemaciclib in non–small cell lung cancer (NSCLC) cells in vitro repair as measured by gH2AX. Mechanistically, abemaciclib and in vivo. inhibited RB phosphorylation, leading to cell-cycle arrest. It also Experimental Design: IR enhancement by abemaciclib in a inhibited mTOR signaling and reduced intracellular amino acid variety of NSCLC cell lines was assessed by in vitro clonogenic pools, causing nutrient stress. In vivo, abemaciclib, when admin- assay, flow cytometry, and target inhibition verified by immu- istered in an adjuvant setting for the second week after fraction- noblotting. IR-induced DNA damage repair was evaluated by ated IR, further inhibited vasculogenesis and tumor regrowth, gH2AX analysis. Global metabolic alterations by abemaciclib with sustained inhibition of RB/E2F activity, mTOR pathway, and IR combination were evaluated by LC/MS mass spectrom- and HIF-1 expression. In summary, our study signifies inhibiting etry and YSI bioanalyzer. Effects of abemaciclib and IR com- the CDK4/6 pathway by abemaciclib in combination with IR as a bination in vivo were studied by xenograft tumor regrowth promising therapeutic strategy to treat NSCLC. delay, xenograft lysate immunoblotting, and tissue section Conclusions: Abemaciclib in combination with IR enhances immunohistochemistry. NSCLC radiosensitivity in preclinical models, potentially provid- Results: Abemaciclib enhanced the radiosensitivity of NSCLC ing a novel biomarker-driven combination therapeutic strategy cells independent of RAS or EGFR status. Enhancement of radio- for patients with NSCLC. Clin Cancer Res; 1–12. 2018 AACR.

Introduction gulated pathways by small molecule tyrosine kinase inhibitors (TKI) or receptor monoclonal antibodies (7–9). Although EGFR- Over the last decade, advances in molecular translational TKIs have been useful in the treatment of EGFR-mutant NSCLC, research have heralded major breakthroughs in the understand- most responses have not proved to be durable with many ing, diagnosis, and management of lung cancer, particularly for patients progressing after 7 to 12 months (4, 8, 10). The most the more common (80%) non–small cell lung cancer frequent mechanism (50%) is concurrent acquisition of novel (NSCLC). NSCLC is subclassified by histology and driver muta- mutation in exon 20 of EGFR, encoding for T790M making tions such as mutated KRAS and activating mutations in the tumors refractory to the existing EGFR-TKI therapy (8, 11). Apart epidermal growth factor receptor (EGFR) tyrosine kinase (TK) from EGFR-TKI, radiotherapy either alone or in combination domain (1–4). The two most common EGFR-TK domain muta- with chemotherapy, remains the primary modality of treatment tions are exon 19 deletions (60%) and L858R missense sub- for patients with stage III NSCLC. For stage I and II NSCLC, stitutions resulting in constitutive activation of the receptor radiotherapy is an alternative curative option to surgery for without ligand binding (3, 5, 6). Constitutive activation of patients who are medically inoperable or refuse surgery. Overall receptors or protein kinases stimulates a complex cascade of radiotherapy is an important palliative treatment modality to cross-signaling pathways leading to uncontrolled growth, pro- treat symptoms from the primary or bone or brain metastases liferation, and survival (2, 3). Successful targeted therapies in and improve patients' quality of life (12). Despite these medical NSCLC involve the identification and inhibition of these upre- interventions, 5-year survival rates of NSCLC patients are less than 5% (4, 13). Hence, there is an urgent need to target other signaling pathways or design combination therapy that is more Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, effective than first-line single agents while balancing toxicity and Bethesda, Maryland. costs. Other than the EGFR or MEK/ERK pathway, cyclin D Note: Supplementary data for this article are available at Clinical Cancer kinase 4/6 (CDK4/6) activity is typically deregulated and over- Research Online (http://clincancerres.aacrjournals.org/). active in various cancers including NSCLC (14, 15). Corresponding Author: James B. Mitchell, Center for Cancer Research, National CDK4 and CDK6 are cyclin-dependent kinases that control the Cancer Institute, Building 10, Room B3-B69, 9000 Rockville Pike, Bethesda, MD transition between the G1 and S-phase of the cell cycle. A major 20892. Phone: 240-858-3101; Fax: 240-541-4528; E-mail: [email protected] target of CDK4 and CDK6 during cell-cycle progression is the doi: 10.1158/1078-0432.CCR-17-3575 retinoblastoma protein (RB). When RB is phosphorylated, its 2018 American Association for Cancer Research. growth-suppressive properties are inactivated. Selective CDK4/6

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All cell lines were authenticated within the past 6 months by Translational Relevance IDEXX Bioresearch using Cell Check 9 [9 allele marker STR (short- Radiotherapy plays a significant role in the management of tandem repeat) profile and interspecies contamination test; Sup- non–small cell lung cancer (NSCLC). It is useful for all stages plementary File S1]. All cells were cultured in RPMI-1640 (Invi- of disease with or without additional treatment modalities, trogen), supplemented with 10% FBS and 1% penicillin/strepto- such as surgery and chemotherapy. However, the response rate mycin at 37 C in a humidified 5% CO2 atmosphere. All CDK4/6 of some tumors remains problematic. The present study inhibitors were purchased from Selleck Chemicals. A stock solu- evaluates the combination of a highly specific CDK4/6 inhib- tion of drug was prepared in 100% dimethyl sulfoxide (Sigma- itor, abemaciclib, with ionizing radiation (IR) to enhance Aldrich) and stored at 70C. The drug was diluted in fresh media radiation sensitivity of NSCLC in vitro and in vivo. Our data prior to each experiment. Abemaciclib was added to cells for indicate that this novel combination efficiently radiosensi- 24 hours after IR treatment at a concentration of 10 mmol/L. tized proliferative and plateau-phase tumor cells and tumor Clonogenic cell survival studies were performed as described xenografts with minimal normal cell radiosensitization. Abe- before (24, 25). Experiments were repeated three independent maciclib inhibited IR-induced DNA damage repair and caused times. Radiation dose-modifying factors (DMF) were determined RB-dependent cell-cycle arrest. Further, the study identified at 10% survival levels by dividing the radiation dose for control by possible predictive biomarkers (p53, RB, and SDF-1) to guide the radiation dose for drug treated. DMFs > 1.0 indicate enhance- treatment response and efficacy of the combination. Collec- ment of radiosensitivity. tively, this study highlights the CDK4/6 axis as a potential radiation target for NSCLC and warrants assessment of abe- Immunoblotting maciclib in clinical trials as a radiation modifier. Exponentially growing cells were washed twice with chilled PBS and cell pellet were collected as a function of time following irradiation. Total protein extraction from cultured cells and xenograft tumors were performed as previously described (25). inhibitors "turn off" these kinases and dephosphorylate RB, Acid soluble histone proteins for gH2AX were extracted in resulting in a block of cell-cycle progression in mid-G prevent- 0.2 mol/L sulfuric acid. Protein concentrations were deter- 1 fi ing proliferation of cancer cells (16). Recent studies have mined using BCA reagent (Thermo Fisher Scienti c). Protein identified poor responses to EGFR-TKIs in lung adenocarcino- samples were resolved on SDS-PAGE and immunoblotting was ma patients with the altered CDK4/6-RB pathway (17, 18). In performed as described before (24, 25). Details of antibodies preclinical mouse models, KRAS-driven lung cancer is highly used for immunoblotting are described in Supplementary dependent on CDK4, and genetic or pharmacologic ablation of Experimental Procedures. CDK4 activity has effects on both the establishment and main- fi tenance of these tumors (19, 20). Palbociclib (PD0332991), a ATP and NAD quanti cation selective CDK4/6 inhibitor, has been shown to sensitize lung ATP (catalog no. K354-100) and NAD (catalog no. K337) fl cancer cells to EGFR-TKI, gefitinib (21). In addition, the MEK colorimetric assays were performed using BioVision kit. Brie y, 6 inhibitor (trametinib) in combination with palbociclib has 1 10 cells were treated with abemaciclib after IR exposure. significant anti-KRAS-mutant NSCLC activity and radiosensitiz- Samples were collected 24 hours post-IR, and the assays ing effects in preclinical models (22). Silencing CDK4 in breast were performed according to the manufacturer's instructions. cancer cells leads to enhanced radiosensitivity, indicating tar- Experiments were performed in triplicate. geting the CDK4/6-cyclin D axis is a rational approach for enhancing tumor IR response (23). Currently, there are three Metabolite extraction and mass spectrometry analysis highly selective CDK4/6 inhibitors (palbociclib, ribociclib, and Cells treated with abemaciclib post-IR for 24 hours were abemaciclib) approved by FDA for hormone receptor positive collected by centrifugation and washed with 5% mannitol and Her2 negative breast cancer patients. Currently, several solution, treated with methanol and water containing internal clinical trials are underway to evaluate these agents as mono- standards (H3304-1002, Human Metabolome Technologies). therapy or combination therapy for various solid tumors. In the Samples were incubated on ice for 5 minutes with occasional present study, we investigated the effects of abemaciclib alone mixing. The extract was centrifuged at 2,300 g and 4 Cfor5 fi and in combination with IR on NSCLC in vitro and in vivo. minutes and centrifugally ltered through a Millipore 5-kDa fi In summary, our results demonstrate abemaciclib as a novel cutoff lter at 9,100 gand4C for 120 minutes to remove fi radiosensitizer for NSCLC in vitro and in vivo. This study also proteins. The ltrate was centrifugally concentrated, dried, and identified potential predictive biomarkers of response to this resuspended in 50 mL of Milli-Q water for capillary electro- novel combinatorial therapy. Mechanistically, abemaciclib radio- phoresis mass spectrometry (CE-MS) analysis. A detailed expla- sensitizing effect includes IR-induced DNA damage repair inhi- nation of CE-TOF and QqQMS methods can be found in bition, sustained inhibition of PI3K/mTOR signaling, amino acid Supplementary Experimental Procedures. starvation leading to enhanced cell death in vitro, and inhibition of tumor vasculogenesis post-IR in vivo. Xenograft studies All animal experiments were carried out in accordance with protocols approved by the National Cancer Institute's Animal Materials and Methods Care and Use Committee (ACUC). For radiation regrowth delay Cell survival studies studies, tumor cells (1 106) were injected into the subcutaneous Human lung cancer cell lines (H1975, H820, H1299, H1650, space of the right hind leg of athymic nude mice, 5 to 6 weeks of H82, H460, A549) and MCF-10A were purchased from ATCC. age, bred at the National Cancer Institute's Animal Production

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Area (Frederick, MD). Individual mice were ear tagged and ran- exposure alone (27). H82 cells, lacking functional RB protein, domized on day 6 of tumor growth into the following 4 groups showed no radiosensitization by abemaciclib (Fig. 1B). Impor- (8 mice/group): vehicle control, vehicle control þ fractionated IR, tantly, the normal mammary epithelial cell line MCF-10A abemaciclib control, and abemaciclib þ fractionated IR. Fraction- was not radiosensitized by abemaciclib with a DMF of ated local IR (2 Gy/fraction) was delivered on Monday, Wednes- 1.03 0.12 (Fig. 1C). All data described to this point apply day, and Friday for a total dose of 6 Gy. Abemaciclib (100 mg/kg) to exponentially growing cells. Because tumors often have a was delivered daily (Monday–Friday) by oral gavage for either 1 or large fraction of nonproliferating cells, studies combining abe- 2 weeks starting at day 6. Three additional mice per group were maciclib and IR exposure were evaluated using quiescent/pla- treated in parallel for tumor harvest. teau phase H460 and H460DNp53 cells. Figure 1D shows that plateau phase H460 cells were still sensitive to abemaciblib and IR although with a reduced DMF of 1.35 0.15, whereas Statistical analysis H460DNp53 plateau phase cells did not respond to abemaci- fi fi Statistical signi cance was quanti ed using ImageJ and clib chemoradiation (DMF, 1.04 0.01;Fig.1D).Collectively, fi GraphPad Prism 7 software. Quanti cation of xenograft group our data indicate abemaciclib as a novel radiation modifier for t growth differences was assessed using the Student test in NSCLC cells, independent of mutated EGFR and KRAS, but Microsoft Excel. dependent on a functional RB and possibly on a functional p53 protein status. Results Abemaciclib enhances radiosensitivity of NSCLC cells in vitro Abemaciclib significantly inhibits radiation-induced DNA To determine the radiation enhancement ability of three repair with post-IR treatment novel CDK4/6 inhibitors (palbociclib, ribociclib, and abema- We further investigated the influence of abemaciclib on IR- ciclib; 24-hour exposure to 1 mmol/L and 10 mmol/L with single induced DNA damage repair by assessment of gH2AX IR dose), pilot studies were performed in H460 cells both with (at Ser139) induction and the time-dependent resolution which pre- and post-IR treatment protocols (Supplementary Fig. S1A– is indicative of DNA double-strand break repair (28). Fig. 2A S1C). Abemaciclib at a concentration of 10 mmol/L showed shows gH2AX kinetics following 7.5 Gy and abemaciclib treat- enhanced radiation sensitivity of H460 cells with single IR dose ment in H460 and H1299 cells. Following 7.5 Gy gH2AX (Supplementary Fig. S1C). Abemaciclib failed to enhance IR rapidly increased in H460 (7.9-fold at 0.5 hour) and H1299 cell killing with DMF of 1.03 (0.03; Supplementary Fig. S1D) (6.7-fold at 0.5 hour) cells, but resolved to unirradiated control when administered 24 hours prior to IR treatment. Palbociclib levels (1.1- and 1.2-fold, respectively) 24 hours post-IR. With and ribociclib at 1 mmol/L or 10 mmol/L concentration failed to post-IR abemaciclib treatment incubation, gH2AX remained exhibit enhancement of radiation sensitivity in H460 cells elevated in H460 (3.92-fold at 24 hours) compared with when added either 24-hour pre- or post-IR treatment (Supple- untreated control (Fig. 2A) indicating inhibition of DNA dam- mentary Fig. S1A and S1B). We further extended our pilot age repair. However, H1299 cells treated with abemaciclib post- observation of combining IR and abemaciclib (24-hour post- IR resulted in similar gH2AX repair kinetics to IR alone treated IR treatment) across a panel of lung cancer cell lines with varied cells over a 24-hour period, which is consistent with lack of genetic background (Table 1; ref. 26). Abemaciclib (10 mmol/L) abemaciclib radiosensitization in this cell line (Fig. 2A). To when added for 24-hour post-IR enhanced radiosensitivity for further evaluate the impact of abemaciclib on DNA damage majority of NSCLC cell lines (Fig. 1A) with DMF ranging repair, expression of key DNA repair proteins such as Rad51, between 1.3 and 1.71. Abemaciclib toxicity and DMF for each phosphorylated Chk2, and ATM following abemaciclib cell line are shown in Table 1. Interestingly, cells deficient for or abemaciclib þ IR were observed to be reduced in H460 functional p53 (H460DNp53, H1299, and H1650) were non- cellswhencomparedwithuntreated control (Fig. 2B and C). A responsive to abemaciclib þ IR combination (Fig. 1B). How- reduction of phosphorylated ATR (, P < 0.005) and DNA-PKc ever, H1975 cells, which does have a mutated p53, was highly (, P < 0.005) with abemaciclib and IR combination sensitive to abemaciclib incubation (Fig. 1A), and likewise to IR also suggested a significant DNA-DSB repair inhibition.

Table 1. Summary of abemaciclib's effect on NSCLC cell line toxicity, cell cycle, and radiation sensitivity Mutational status Cell cycle Radiation effect Toxicity Cell lines Histology EGFR RB1 KRAS TP53 p16 PTEN % G1 % S % G2 DMF(SD) PE (%) H460 LCC DEL? WT MUT WT DEL WT 78 (66) 4 13 1.71 (0.15) 22 H460 DN p53 LCC WT WT MUT DN DEL WT 79 (61) 11 10 1.14 (0.02) 50 A549 NSCLC WT WT MUT WT DEL WT 90 (72) 8 2 1.43 (0.02) 60 H1299 NSCLC WT WT WT DEL WT WT 77 (55) 13 9 1.01 (0.01) 85 H1975 NSCLC MUT WT WT MUT WT WT 69 (49) 10 21 1.63 (0.02) 54 H820 NSCLC MUT WT MUT MUT WT WT 82 (68) 15 3 1.3 (0.51) 66 H1650 NSCLC MUT WT WT MUT DEL / 64 (51) 16 20 1.0 (0.03) 56 H82 SCLC WT / WT WT WT WT 51 (49) 20 29 1.02 (0.03) 77 MCF10A Normal breast Amp WT WT MUT DEL WT 87 (49) 5 8 1.03 (0.12) 59 H460 plateau LCC WT WT MUT WT DEL WT 94 (92) 2 4 1.35 (0.15) 33 H460 DN p53 plateau LCC WT WT MUT DN DEL WT 89 (79) 7 4 1.04 (0.01) 49 NOTE: Values in parentheses are the % G1 of untreated cells. MCF710A are immortalized normal breast epithelial cells. Mutational status of most cell lines determined based on Blanco and colleagues (26) and Molina-Vila and colleagues (48). Toxicity was based on a 10 mmol/L abemaciclib exposure for 24 hours. Abbreviations: Amp, gene ampliﬁcation; DMF, dose-modifying factor; DN, dominant negative gene; LCC, large cell carcinoma; MUT, Mutated gene; NSCLC, non–small cell lung cancer; PE, plating efﬁciency; SCLC, small cell lung cancer; SD, standard deviation; WT, wild-type gene; (/) represents null status for gene.

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Importantly, no significant changes in the expression of the hours as compared with a 4-hour drug exposure. Reduced RB above DNA-DSB repair proteins were observed for the p53- phosphorylation post abemaciclib was followed by a concom- deficient cell line, H1299 (Fig. 2D and Supplementary itant increase in p21 expression (Fig. 2E and Supplementary Fig. S2A). We confirmed the inhibition of RB phosphorylation Fig. S2B) leading to an increased G1 cell-cycle arrest (Table 1). at Ser780 in H460 (Fig. 2E) and H1975 cells (Supplementary Abemaciclib incubation also reduced the expression of RB/ 811/807 Fig. S2B) as well as at Ser (Supplementary Fig. S2C) in E2F target proteins such as TopoIIa (marker for G1–S H460 cells after 10 mmol/L of abemaciclib treatment for 24 transition; Fig. 2E and Supplementary Fig. S2B) and showed

Figure 1. Radiation survival curves for lung cancer cell lines treated with abemaciclib. A–D, All cells were treated with abemaciclib (10 mmol/L, dashed curve with closed circles) or with DMSO control (0.1%, solid curve with open circles) for 24 hours immediately after irradiation and clonogenic assay were performed. Data are representative of 2 to 3 independent experiments. DMFs, abemaciclib toxicities, its effect on cell cycle, tumor histology, and mutational status of indicated cell lines are shown in Table 1.

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Figure 3. Abemaciclib inhibits mTOR signaling in H460 cells. A, Immunoblot showing reduced expression of downstream mTOR effectors p-S6, p-AKT, p-4EBP1, p-P70S6K, and p-RAS40 (dilution, 1:1,000) after abemaciclib treatment in the presence or absence of IR. B and C, Colorometric assay measuring levels of ATP (B) and total NAD (C) levels in H460 cell lysates obtained after treatment with or without IR and abemaciclib. Data are represented as pmol/L of ATP normalized to total cell number. Error bar, mean SEM and statistical signiﬁcance: , P < 0.01; , P < 0.001.

asignificant decrease in p-HH3 (histone H3) expression at Abemaciclib inhibits mTOR signaling in vitro Ser 10 (marker for mitosis) as observed by flow cytometry Another signaling pathway known to influence IR sensitivity (Supplementary Fig. S2D). Significantly, the G1 block after is the mTOR pathway that also regulates tumor growth, tumor abemaciclib incubation was not observed in the RB-deficient cell metabolism, and cell-stress responses (29, 30). Multiple H82cellline(Table1).Therewasalsoanaccumulationoflate marker proteins of mTOR activation were clearly reduced by apoptotic cells (annexin V positive þ PI positive) after 24 hours abemaciclib incubation in H460 cells (Fig. 3A). A reduction in of abemaciclib and IR treatment in H460, but not in H1299 expression of p-Akt (Ser 473), p-S6, p-4EBP1, and p-p70S6K and H460DNp53 (Supplementary Fig. S2E) cells. Taken togeth- (Fig. 3A) was observed. Similar observations were made in er, our results suggest that inhibition of CDK4/6 activity by H1975 cells (data not shown). mTOR also regulates ATP and abemaciclib in NSCLC cells causes enhanced G1 arrest and total NAD levels (29, 30) as shown in Fig. 3B and C, respec- inhibits DNA–DSBs repair pathways leading to enhanced IR tively. ATP and total NAD levels in H460 cells were reduced by sensitization. 35% and 29% by abemaciclib and IR exposure compared with

Figure 2. Effect of abemaciclib on radiation induced DNA damage repair and the CDK4/6-RB pathway in NSCLC cells. A, Western blot analysis of phosphorylated gH2AX in isolated histones from H460 and H1299 cell lines as a function of time following treatment with abemaciclib (10 mmol/L) and IR (7.5 Gy). Individual experiment densitometry results were normalized based on the corresponding lane H2A-loading control, and values beneath each row indicate fold change (FC) derived from 3 independent experiments. B, Immunoblot showing expression of DNA damage repair pathway proteins in H460 cells as function of time following treatment with abemaciclib þ IR (7.5 Gy) combination. C and D, Densitometry analysis of three independent experiments in H460 cells (C) and H1299 cells (D) after IR and abemaciclib treatment. The expression of p-ATR, p-ATM, and p-Chk2 was normalized based on their corresponding total protein expression. Error bar, mean SEM from three independent experiments. Statistical signiﬁcance is represented as , P < 0.05; , P < 0.005; and , P < 0.0005 over DMSO control. E, Immunoblot showing expression of key proteins in the CDK4/6-RB axis in H460 cells following treatment with abemaciclib alone or in combination with IR.

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Figure 4. Abemaciclib triggers nutrient stress in H460 cells by altering metabolism. A, LC/MS mass spectrometry analysis showing fold expression of steady- state levels of total NEAA (nonessential amino acid) and EAA (essential amino acid) in H460 cells treated in the presence and absence of abemaciclib or IR (5 Gy). The concentrations of metabolites are expressed as fold change. B and C, Levels of glucose and glutamine consumption and glutamine and lactate secretion were measured by bioanalyzer. The levels of indicated metabolites in the supernatants were normalized to concentration (mg) of protein in the sample. Experiments were done in triplicate; error bar, mean SD. , P < 0.01; , P < 0.001; and , P < 0.0001. D, Immunoﬂuorescence analysis of H460 cells treated with DMSO or abemaciclib for 24 hours, showing expression of Glut-1 (glucose transporter, shown in green plots A and B, respectively) and ASCT2 (amino acid transporter, shown in red plots C and D, respectively). DAPI was used to stain nucleus (shown in blue). Images were taken at 60 using Zeiss 780 confocal microscope. Scale bar, 10 mm.

IR only, respectively (Fig. 3B and C). Reduction in ATP levels levels thereby rendering NSCLC cells vulnerable to enhanced by abemaciclib is important as chromatin-remodeling com- radiosensitization. plexes, which are ATP dependent play an important role in all three major DNA repair pathways: nucleotide excision repair, Abemaciclib alters NSCLC metabolism by disrupting key homologous recombination, and nonhomologous end join- nutrient sensing pathways ing (31). Reduced intracellular ATP by abemaciclib may play a In recent years, it has been well appreciated that cancer cells role in inhibition of IR-induced DNA damage repair thereby have the ability to rewire their metabolism and energy production causing enhanced radiosensitivity. Collectively, these results networks in response to altered microenvironment conditions to suggest that abemaciclib-mediated inhibition of mTOR could support rapid proliferation, invasion, metastasis, and resistance to possibly lead to reduced intracellular ATP and total NAD cancer treatment (32, 33). mTOR remains central in regulating

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tumor metabolism (29, 30). Our study indicated sustained mTOR Abemaciclib inhibits IR-induced vasculogenesis and tumor inhibition by abemaciclib and abemaciclib þ IR combination regrowth in combination with fractionated IR (Fig. 3A). Hence, we next evaluated the effects of abemaciclib þ IR A pilot H460 xenograft study was conducted combining combination in NSCLC on tumor cell metabolism. Steady-state fractionated IR (three 2 Gy fractions) with daily abemaciclib levels of 116 metabolites in H460 cells using targeted capillary (100 mg/kg) treatment through oral gavage. Initially, two daily electrophoresis time of flight (CE-TOF) and tandem triple qua- abemaciclib doses (immediately after radiation and 6 hours later) druple mass spectrometry (QqQMS) was determined. Glycolytic were delivered immediately after the first IR fraction and the intermediates such as fructose 1, 6 bisphosphate were elevated (, second day; however, this was not well tolerated and subsequently P < 0.005) after 24 hours of abemaciclib treatment, whereas abemaciclib was given as daily single dose for the remainder of the downstream intermediates such as 2-PG (2-phosphoglycerate) week (Wednesday–Friday). This regimen of abemaciclib treat- and 2-PEP (2-phosphoenylpyruvate) were reduced (, P < 0.005 ment resulted in modest radiosensitization (Supplementary Fig. and , P < 0.05 , respectively) suggesting that abemaciclib altered S4A) and an increased delay in the tumor regrowth kinetics (P ¼ the activity of glyceraldehyde 6 phosphate dehydrogenase 0.02) as compared with radiation only control, followed with (G6PD). With exception of reduced levels of citrate, most TCA rapid regrowth. In an attempt to further enhance tumor regrowth cycle metabolites did not show any significant change after delay, we considered the tumor vasculature. Targeting tumor abemaciclib treatment (Supplementary Fig. S3A). Interestingly, vasculature has long been considered for both radiation and abemaciclib caused a significant decrease in steady-state levels chemoradiation as a means of tumor destruction (37). The novel of both nonessential amino acid (NEAA) and essential amino finding of Brown and colleagues who demonstrated using a brain acids (EAA; , P < 0.0001), suggesting a possible amino acid tumor xenograft model, that tumor vasculature can recover post- deficiency (nutrient stress) in H460 cells (Fig. 4A and Supple- IR by colonization of circulating BMDC (bone marrow derived mentary Fig. S3B). One of the best-studied ways by which cells cells) primarily CD11bþ myeloid cells. This pathway of blood maintain their amino acid levels during starvation or nutrient vessel formation, secondary to tumor angiogenesis which is stress in vitro is by elevating a general amino acid control inhibited by IR (38), known as vasculogenesis, restores tumor (GAAC) pathway (34). Studies have indicated that glutamine vasculature facilitating recurrence of tumor following radiation depletion can also trigger the GAAC pathway, by elevating (39, 40). They further showed that the stimulus for the influx of þ expression of amino acid transporters such as ASCT2, thereby CD11b cells into tumors following IR is increased levels of increasing amino acid uptake and elevating intracellular amino hypoxia (and HIF-1 expression) that drives secretion of the acid levels, such as that of leucine (Leu). Increased Leu has been chemokine stromal cell-derived factor-1 (SDF-1). The kinetics of showntoreactivatemTOR(35,36).Interestingly,inour vasculogenesis in the brain tumor model was observed to be as metabolic analysis, abemaciclib significantly downregulated early as 2 weeks postradiation leading to enhanced tumor levels of Leu (Supplementary Fig. S3B). In addition to changes regrowth (39). Hence, we hypothesized that should fractionated in steady-state levels of glycolytic and TCA-cycle metabolites, IR during the 1st week of treatment initiate vasculogenesis, then we also observed the kinetics of glucose and glutamine uptake an additional week of adjuvant abemaciclib treatment might by H460 cells using a YSI-Bioanalyzer. In the presence of provide additional tumor regrowth delay. abemaciclib, both glucose and glutamine uptakes were To test this hypothesis, we conducted a second in vivo study decreased (Fig. 4B and C). In agreement with the observed where mice with H460 xenografts were exposed to fractionated IR decrease in Leu, glutamine, and glucose uptake, abemaciclib (2 Gy) on Monday, Wednesday, and Friday in addition to daily treatment altered the surface distribution of both ASCT2 gavage of the abemaciclib (100 mg/kg) immediately after IR for 5 (shown in red, plot D) and glucose transporter (GLUT1, shown days in a week (1st week) followed by an additional five daily in green panel, plot B; Fig. 4D). Other surface proteins, such as treatments of abemaciclib only without IR (second week). In the CD98 (also known as 4F2HC or SLC3A2, neutral amino acid absence of radiation, abemaciclib displayed a modest effect on transporter) and CD147, a chaperone protein that mediates H460 xenograft regrowth by 2 days (P ¼ 0.14) versus control transport of MCT1 and MCT4 to the plasma membrane, were untreated tumors (Fig. 5A). However, abemaciclib in combina- not affected. Collectively, abemaciclib treatment reduced ATP tion with IR showed synergistic delay in tumor regrowth of 8 and 9 levels and restricted access to key metabolic substrates mainly days, respectively as compared with IR only and control tumor amino acids, glutamine, and glucose possibly leading to nutri- (P ¼ 0.006 and P ¼ 0.001, respectively). Adding a second week of ent and energy stress. These results indicate that abemaciclib adjuvant abemaciclib treatment enhanced tumor delay compared exposure leads to sustained mTOR inhibition, limits access to with 1 week of treatment (Supplementary Fig. S4A). This study extracellular amino acid pools, and decreases intracellular ATP was repeated with similar findings as shown in (Supplementary levels thereby facilitating NSCLC to become sensitive to IR. Fig. S4C). We next evaluated tumor tissue at the end of the second

Figure 5. Effect of abemaciclib and fractionated radiation on tumor regrowth and radiation induced vasculogenesis. A and B, H460 xenograft showing enhanced tumor regrowth delay by daily administration of 100 mg/kg body weight of abemaciclib through oral gavage for 5 days immediately after 2 Gy 3 IR dose administered every alternate day, indicated as week 1. The drug dosing was continued for another 5 days, indicated as week 2. A, The significance for tumor regrowth delay was measured when tumor size reached 800 mm3. The statistical significance is derived from 8 animals per group. B, Confocal microscopy showing expression of Ki-67, p-HH3, and p-RB and infiltration of BMDC (bone marrow derived cells) such as CD45, CD11bþ monocytes, and F4/80þ macrophages along with SDF-1 secretion in frozen section of H460 xenograft derived from week 2 treatment of abemaciclib with fractionated radiation. Nucleus is stained with DAPI (shown in blue). All images were taken at a magnification of 100. Scale bar, 10 mm. C, Mean fluorescence intensity (MFI) of immunopositive cells of indicated proteins was measured for approximately eight random fields of the immunostained section using ImageJ. The fold expression was calculated by normalizing the MFI to that of MFI of DMSO-treated tumor section. Error bar represents mean SD. , P < 0.001; , P < 0.0001. D, Immunoblot analysis showing expression of HIF1a in H460 xenograft for week 1 and week 2 of abemaciclib administration after fractionated radiation.

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week of adjuvant treatment to determine if IR-induced vasculo- patient populations for better therapeutic outcome. While previ- genesis occurred and if abemaciclib inhibited the induction. ous in vitro studies suggested RB, cyclin D, and p16 could predict Reduced immunohistochemical staining of fluorescently labeled the response to palbociclib (45), results from phase II/III trials þ CD11b monocytes, SDF-1, CD45þ, and F4/80-positive macro- showed no significant correlation between drug response and the phages were observed in week 2 tumors following abemaciclib expression of p16 (45), Ki-67, cyclin D1 amplification (46), and IR þ abemaciclib combination treatment. In good agreement PIK3CA or ESR1 (47) mutational status, leaving no established with Brown and colleagues, week 1 tumor post-IR treatment did prognostic or predictive biomarkers. þ not show enhanced SDF-1 (40) and CD11b BMDC infiltration The preclinical findings of the current study provide the basis (data not shown). Abemaciclib alone inhibited RB phosphory- for future clinical trials that are biomarker integrated in NSCLC by lation and tumor cell proliferation as seen by reduced p-HH3 and combining abemaciclib with IR. Clonogenic cell survival data Ki-67 staining (Fig. 5B and C). Inhibition of vasculogenesis in across a panel of cell lines harboring genotypic mutations in key week 2 tumors by abemaciclib and IR treatment was quantified driver proteins involved in the progression of NSCLC such as and shown in Fig. 5C. Western analysis of tumor lysates confirmed mutation in KRAS, EGFR-TK domain, p53, RB, and p16 indicated modest RB inhibition as seen by cyclin A expression in abema- that abemaciclib when administered immediately post-IR pri- ciclib alone and combination with IR in week 1 tumors (Supple- marily radiosensitized cells with functional p53 and RB proteins, mentary Fig. S4B). In good agreement with reduced p-RB expres- but was independent of the mutational status of EGFR, KRAS, and sion in week 2 tumor sections (Fig. 5C), we also observed a p16 protein (Fig. 1 and Table 1). Interestingly, H1975 cells were sustained inhibition of cyclin A expression upon abemaciclib and radiosensitized by abemaciclib (Fig. 1A) but have a mutated p53 abemaciclib þ IR combination in week 2 tumors (Supplementary (Table 1; ref. 48). It has been reported for NSCLC that certain p53 Fig. S4B). These results clearly show that inhibition of the CDK4/6 mutations can be nondisruptive leading to a Gain-of-function pathway leads to inhibition of IR-induced tumor vasculogenesis. (GOF) and patients with these mutations have poorer prognosis Furthermore, in H460 xenografts, abemaciclib treatment led to a than patients with disruptive p53 mutations (48). H1975 cells sustained inhibition of p-S6 levels indicating reduced mTOR were identified as having a SNP in exon 8 (R273H) which was activation (Supplementary Fig. S4B). IR treatment enhanced found to be nondisruptive (48). The H1975 cells used in this expression of HIF-1 at week 1, was dramatically inhibited by study was also found to have the R273H mutation through whole- abemaciclib (Fig. 5D). Further, abemaciclib in an adjuvant setting exon sequencing (data not shown) thus suggesting a possible also inhibited IR-induced induction of HIF-1 at week 2, but not explanation for our IR sensitization results. Additional research is completely (Fig. 5D). Abemaciclib inhibited HIF-1 expression certainly needed to clarify this observation that in some cases independent of IR in untreated tumors (week 2) as tumor growth abemaciclib can radiosensitize p53-mutated cells. enhanced the hypoxic fraction (Fig. 5D). The mechanism of how Radiosensitization of H460 xenografts using fractionated radi- abemaciclib inhibits induction of HIF-1 in vivo remains to be ation (Fig. 5), combined with abemaciclib, indicated that CDK4/6 established and will be a topic of further studies. inhibition may be an excellent target for clinical radiotherapy. Our data attribute the radiosensitization effect of abemaciclib to several parallel mechanisms in vitro and in vivo. In vitro, abema- Discussion ciclib and IR combination resulted in significant inhibition of IR- With recent development and FDA approval of highly specific induced DNA damage repair as assessed by gH2AX levels and CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib) for compromised activation of key DNA damage response proteins advanced metastatic breast cancer, there has been growing interest to repair DNA (Fig. 2). In the present study, abemaciclib at in design of multiple clinical trials with these agents in various 10 mmol/L for 24 hours leads to sustained inhibition of mTOR solid tumors (https://clinicaltrials.gov/). Successful phase I trial of both in vitro and in vivo (Fig. 3A and Supplementary Fig. S4B). abemaciclib in patients with advanced stage NSCLC and other mTOR inhibition by abemaciclib further leads to reduced amino solid tumors is very encouraging regarding the design of future acid pools as seen by global metabolic profiling (Fig. 4A). These combination therapy (20). However, recent reports of abemaci- alterations in the nutrient and energy status of cells possibly clib failure as monotherapy in phase III trial for KRAS-mutant increased activated stress responses which lead to cell death via NSCLC have been discouraging (http://www.ascopost.com/ induction of apoptosis in vitro. Clearly, abemaciclib affects mTOR News/58135). There are, nonetheless, potential combinatorial signaling resulting in reduction in ATP levels and other essential therapies where CDK4/6 inhibitors may be beneficial. Preclinical metabolite which may interfere with IR-induced DNA damage studies have indicated that palbociclib can sensitize KRAS-mutant repair. However, the mechanism of mTOR inhibition by abema- NSCLC cells to MEK inhibitor and EGFR-TKI (22). Ribociclib was ciclib is unclear and requires additional studies. shown to sensitize PIK3CA-mutant breast cancer to PI3K inhibi- Most interestingly, our study implicates a unique role of tors (41). Preclinical studies have shown palbociclib to modify IR CDK4/6 inhibition in IR-induced vasculogenesis in vivo as a response in breast cancer (42), glioblastoma (43), and medullo- contributor to the enhanced IR response. Inhibition of IR-induced blastoma (44). Our preclinical data strongly support abemaciclib, vasculogenesis by abemaciclib is in good agreement with the as a potent radiation modifier in NSCLC, whereas our pilot studies findings of Brown and colleagues, who showed that IR to a tumor indicate that palbociclib and ribociclib did not yield IR enhance- can induce tumor vasculogenesis leading to recurrence in a GBM ment. This could possibly be tumor-type-dependent effect of the xenograft model (39). He further demonstrated that an inhibitor drug, drug concentration, and the IR treatment strategy used in of vasculogenesis (SDF-1/CXCR4 pathway inhibitor, AMD3100) our study. Despite CDK inhibitor successes in managing patients when administered the second week post-IR resulted in enhanced with advanced disease (14), there are still challenges in the tumor regrowth delay (39). Phase I/II trial studies evaluating optimization of CDK inhibitors in clinical practice. A major AMD3100 (plerixafor) after radiation therapy and temozolomide hurdle is lack of predictive biomarkers to screen appropriate in patients with high-grade GBM have shown promising local

OF10 Clin Cancer Res; 2018 Clinical Cancer Research

Enhancement of Radiosensitivity by Abemaciclib in NSCLC

tumor control (49). Our data are consistent with these findings in Authors' Contributions that SDF-1, while not expressed in the tumor samples the 1st week Conception and design: S. Naz, J.A. Cook, J.B. Mitchell of treatment, was enhanced during the second week post-IR (Fig. Development of methodology: S. Naz, R. Choudhuri, J.B. Mitchell 5B and C). Abemaciclib treatment during the second week inhib- Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Naz, R. Choudhuri, M. Wissler, J. Gamson, ited SDF-1 induction (Fig. 5B and C), thus inhibiting vasculogen- A. Mathias esis. In addition to inhibiting IR-mediated vasculogenesis (Fig. 5) Analysis and interpretation of data (e.g., statistical analysis, biostatistics, CDK4/6 inhibition also exerted sustained mTOR inhibition and computational analysis): S. Naz, R. Choudhuri, A. Mathias, J.A. Cook, of HIF-1 in vivo. CDK4/6 treatment also prevented tumor cell entry J.B. Mitchell to mitosis as shown by reduced p-HH3 and Ki-67 staining Writing, review, and/or revision of the manuscript: S. Naz, J.A. Cook, (Fig. 5C). These findings are crucial for radiotherapy because both J.B. Mitchell Administrative, technical, or material support (i.e., reporting or organizing enhanced tumor hypoxia (induction of HIF-1) and mTOR acti- data, constructing databases): S. Naz, A. Sowers, M. Wissler, J.A. Cook vation are well-documented mechanisms for altering radiosensi- Other (performed the clonogenic survival studies): J. Gamson tivity in tumors (24, 50). Collectively, our results suggest that there are multiple mechanisms of abemaciclib radiosensitization Acknowledgments including inhibition of DNA damage repair, mTOR signaling, and The authors thank Lim Langston and Poonam Mannan (NCI Core Imaging IR-induced vasculogenesis. Facility, NIH) for technical assistance on confocal microscopy image capture In summary, the results of the present study suggest that and analysis. abemaciclib and IR combination would be a reasonable way to This research was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, NIH. manage NSCLC patients in early and advance disease settings, with implications on minimizing IR-induced tumor recurrence. fi The costs of publication of this article were defrayed in part by the payment of Our ndings clearly warrant consideration of this novel chemor- page charges. This article must therefore be hereby marked advertisement in adiation combination in clinical trials. accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Disclosure of Potential Conflicts of Interest Received November 29, 2017; revised March 30, 2018; accepted April 27, No potential conflicts of interest were disclosed. 2018; published first May 1, 2018.

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OF12 Clin Cancer Res; 2018 Clinical Cancer Research

Abemaciclib, a Selective CDK4/6 Inhibitor, Enhances the Radiosensitivity of Non−Small Cell Lung Cancer In Vitro and In Vivo

Sarwat Naz, Anastasia Sowers, Rajani Choudhuri, et al.

Clin Cancer Res Published OnlineFirst May 1, 2018.

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