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

Home , CDK inhibitor, Palbociclib

Author Manuscript Published OnlineFirst on May 1, 2018; DOI: 10.1158/1078-0432.CCR-17-3575 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

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, James B. Mitchell*

Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, Bethesda MD

Running Title: Enhancement of radiosensitivity by Abemaciclib in NSCLC

Key words: Abemaciclib, CDK4/6, Radiosensitizer, Metabolism, Vasculogenesis, Cell Cycle NSCLC

Financial support: This research was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, NIH.

* Correspondence should be addressed to:

Dr. James B Mitchell, Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, NIH Building 10, Room B3-B69, 9000 Rockville Pike, Bethesda, MD 20892-1002, USA

Phone: 240-858-3101, Fax: 240-541-4528, Email: [email protected]

Total words: 4560; Figures: 5; Table: 1

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on May 1, 2018; DOI: 10.1158/1078-0432.CCR-17-3575 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translational Relevance (150 words)

Radiotherapy plays a significant role in the management of non–small cell lung cancer

(NSCLC). It is useful for all stages of disease with or without additional treatment modalities such as surgery and chemotherapy. However, the response rate of some tumors remains problematic.

The present study evaluates the combination of a highly specific CDK4/6 inhibitor, Abemaciclib with ionizing radiation (IR) to enhance radiation sensitivity of NSCLC in vitro and in vivo. Our data indicate that this novel combination efficiently radiosensitized proliferative and plateau-phase tumor cells and tumor xenografts with minimal normal cell radiosensitization. Abemaciclib inhibited IR-induced DNA damage repair and caused RB dependent cell cycle arrest. Further, the study identified possible predictive biomarkers (p53, RB and SDF-1) to guide treatment response and efficacy of the combination. Collectively, this study highlights CDK4/6 axis as potential radiation target for NSCLC and warrants assessment of Abemaciclib in clinical trials as a radiation modifier.

Abstract (244 words)

Purpose: To characterize the ionizing radiation (IR) enhancing effects and underlying mechanisms of CDK4/6 inhibitor, Abemaciclib in Non–Small Cell Lung Cancer cells (NSCLC) in vitro and in vivo.

Experimental Design: IR enhancement by Abemaciclib in a variety of NSCLC cell lines was assessed by in vitro clonogenic assay, flow cytometry, and target inhibition verified by immunoblotting. IR-induced DNA damage repair was evaluated by γ-H2AX analysis. Global metabolic alterations by Abemaciclib and IR combination was evaluated by LC/MS mass spectrometry and YSI-Bioanalyzer. Effects of Abemaciclib and IR combination in vivo was studied by xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry.

Results: Abemaciclib enhanced the radiosensitivity of NSCLC cells independent of RAS or EGFR status. Enhancement of radiosensitivity was lost in cell lines deficient for functional p53 and RB protein. Post-IR, Abemaciclib treatment inhibited DNA damage repair as measured by γ-H2AX. Mechanistically, Abemaciclib inhibited RB phosphorylation leading to cell cycle arrest. It also inhibited mTOR signaling and reduced intracellular amino acid pool causing nutrient stress. In vivo, Abemaciclib when administered in an adjuvant setting for the second week post-fractionated IR further inhibited vasculogenesis and tumor re-growth with sustained inhibition of RB/E2F activity, mTOR pathway, and HIF-1 expression. In summary, our study signifies inhibiting CDK4/6 pathway by Abemaciclib in combination with IR as a promising therapeutic strategy to treat NSCLC.

Conclusions: Abemaciclib in combination with IR enhances NSCLC radiosensitivity in preclinical models, potentially providing a novel biomarker driven combination therapeutic strategy for patients with NSCLC.

Introduction

Over the last decade, advances in molecular translational research have heralded major breakthroughs in the understanding, diagnosis and management of lung cancer, particularly for the more common (~80%) Non-Small Cell Lung Cancer (NSCLC). NSCLC is sub-classified by histology and driver mutations such as mutated KRAS and activating mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain (1-4). The two most common EGFR-

TK domain mutations are exon 19 deletions (60%) and L858R missense substitutions resulting in constitutive activation of the receptor without ligand binding (3,5,6). Constitutive activation of receptors or protein kinases stimulate a complex cascade of cross signaling pathways leading to uncontrolled growth, proliferation, and survival (2,3). Successful targeted therapies in NSCLC involves the identification and inhibition of these up-regulated pathways by small molecule tyrosine kinase inhibitors (TKI) or receptor monoclonal antibodies (7-9). Although EGFR-TKIs have been useful in the treatment of EGFR-mutant NSCLC, most responses have not proved to be durable with many patients progressing after 7-12 months (4,8,10). The most frequent mechanism

(~50%) is concurrent acquisition of novel mutation in exon 20 of EGFR, encoding for T790M making tumors refractory to the existing EGFR-TKI therapy (8,11). Apart from EGFR-TKI, radiotherapy either alone or in combination with chemotherapy, remains the primary modality of treatment for patients with stage III NSCLC. For stage I and II NSCLC, radiotherapy is an alternative curative option to surgery for patients who are medically inoperable or refuse surgery.

Overall radiotherapy is an important palliative treatment modality to treat symptoms from the primary or bone or brain metastases and improve patients’ quality of life (12). Despite these medical 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 effective than first line single agents whilst balancing toxicity and costs. Other than EGFR or MEK/ERK pathway cyclin D kinase 4/6 (CDK4/6) activity is typically deregulated and overactive in various cancers including NSCLC (14,15)

CDK4 and CDK6 are cyclin-dependent kinases that control the transition between the G1 and

S phase of the cell cycle. A major target of CDK4 and CDK6 during cell-cycle progression is the retinoblastoma protein (RB). When RB is phosphorylated, its growth-suppressive properties are inactivated. Selective CDK4/6 inhibitors “turn off” these kinases and dephosphorylate RB, resulting in a block of cell-cycle progression in mid-G1 preventing proliferation of cancer cells

(16). Recent studies have identified poor responses to EGFR-TKIs in lung adenocarcinoma patients with altered CDK4/6-RB pathway (17,18). In preclinical mouse models, KRAS-driven lung cancer is highly dependent on CDK4, and genetic or pharmacologic ablation of CDK4 activity has effects on both the establishment and maintenance of these tumors (19,20). Palbociclib

(PD0332991), a selective CDK4/6 inhibitor, has been shown to sensitize lung cancer cells to

EGFR-TKI, gefitinib (21). In addition, the MEK inhibitor (Trametinib) in combination with Palbociclib has significant anti-KRAS-mutant NSCLC activity and radiosensitizing effects in preclinical models (22). Silencing CDK4 in breast cancer cells leads to enhanced radiosensitivity, indicating targeting CDK4/6-CyclinD axis is a rational approach for enhancing tumor IR response (23). Currently, there are three highly selective CDK4/6 inhibitors (Palbociclib,

Ribociclib and Abemaciclib) approved by FDA for hormone receptor positive and Her2 negative breast cancer patients. Currently, several clinical trials are underway to evaluate these agents as monotherapy or combination therapy for various solid tumors. In the present study, we investigated the effects of Abemaciclib alone and in combination with IR on NSCLC in vitro and in vivo.

In summary, our results demonstrate Abemaciclib as a novel radiosensitizer for NSCLC in vitro and in vivo. This study also identified potential predictive biomarkers of response to this novel combinatorial therapy. Mechanistically, Abemaciclib radiosensitizing effect includes IR- induced DNA damage repair inhibition, sustained inhibition of PI3K/mTOR signaling, amino acid starvation leading to enhanced cell death in vitro, and inhibition of tumor vasculogenesis post-IR in vivo.

Materials and Methods

Cell survival studies

Human lung cancer cell lines H1975, H820, H1299, H1650, H82, H460, A549 and MCF-10A were purchased from American Type Culture Collection (ATCC). All cell lines were authenticated within the past 6 months by IDEXX Bioresearch using Cell Check 9 [9 allele marker STR (short tandem repeat) profile and interspecies contamination test] (Supplementary File 1). All cells were cultured in RPMI-1640 (Invitrogen), supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified 5% CO2 atmosphere. All CDK4/6 inhibitors were purchased from Selleck

Chemicals. A stock solution of drug was prepared in 100% dimethyl sulfoxide (Sigma) and stored at −70°C. The drug was diluted in fresh media prior to each experiment. Abemaciclib was added to cells for 24 hours after IR treatment at a concentration of 10 µM. Clonogenic cell survival studies were performed as described before (24,25). Experiments were repeated three independent times. Radiation dose modifying factors (DMFs) were determined at 10% survival levels by dividing the radiation dose for control by the radiation dose for drug treated. DMFs > 1.0 indicate enhancement of radiosensitivity.

Immunoblotting

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). Acid soluble histone proteins for

γ-H2AX were extracted in 0.2 mol/L sulfuric acid. Protein concentrations were determined using

BCA reagent (Thermo Scientific). Protein samples were resolved on SDS-PAGE and immunoblotting was performed as described before (24,25). Details of antibodies used for immunoblotting is described in Supplemental Experimental Procedures.

ATP and NAD Quantification

ATP (Catalog # K354-100) and NAD (Catalog # K337) colorimetric assays were performed using BioVision kit. Briefly, 1x106 cells were treated with Abemaciclib after IR exposure. Samples were collected 24 hours post-IR and the assays were performed according to manufacturer’s instructions. Experiments were performed in triplicates and repeated at least three times.

Metabolite Extraction and Mass Spectrometry Analysis

Cells treated with Abemaciclib post-IR for 24 hours were collected by centrifugation and washed with 5% mannitol solution, treated with methanol and water containing internal standards

(H3304-1002, Human Metabolome Technologies). Samples were incubated on ice for 5 min with occasional mixing. The extract was centrifuged at 2,300 × g and 4°C for 5 min and centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 × g and 4°C for 120 min to remove proteins.

The filtrate was centrifugally concentrated, dried and re-suspended in 50 μL of Milli-Q water for capillary electrophoresis mass spectrometry (CE-MS) analysis. A detailed explanation of CE-TOF and QqQMS methods can be found in Supplemental Experimental Procedures.

Xenograft Studies

All animal experiments were carried out in accordance with protocols approved by the National

Cancer Institute's Animal Care and Use Committee (ACUC). For radiation regrowth delay studies, tumor cells (1x106) were injected into the subcutaneous space of the right hind leg of athymic nude mice, 5 to 6 weeks of age, bred at the National Cancer Institute's Animal Production Area

(Frederick, MD). Individual mice were ear tagged and randomized on day 6 of tumor growth into the following 4 groups (8 mice/group): vehicle control, vehicle control + fractionated IR,

Abemaciclib control, and Abemaciclib + fractionated IR. Fractionated local IR (2 Gy/fraction) was delivered on Monday, Wednesday, and Friday for a total dose of 6 Gy. Abemaciclib (100 mg/kg) was delivered daily (Mon-Fri) by oral gavage for either 1 or 2 weeks starting at day 6. Three additional mice per group were treated in parallel for tumor harvest.

Statistical Analysis

Statistical significance was quantified using ImageJ and GraphPad Prism 7 software.

Quantification of xenograft group growth differences were assessed using the Student t test in

Microsoft Excel.

Results

Abemaciclib enhances radiosensitiviy of NSCLC cells in vitro

To determine the radiation enhancement ability of three novel CDK4/6 inhibitors (Palbociclib,

Ribociclib, and Abemaciclib; 24 hour exposure to 1 µM and 10 µM with single IR dose) pilot studies were performed in H460 cells both with pre- and post-IR treatment protocols

(Supplementary Fig. S1A-C). Abemaciclib at a concentration of 10 µM showed enhanced radiation

sensitivity of H460 cells with single IR dose (Supplementary Fig. S1C). Abemaciclib failed to enhance IR cell killing with DMF of 1.03 (±0.03) (Supplementary Fig. S1D) when administered

24 hours prior to IR treatment. Palbociclib and Ribociclib at 1 µM or 10 µM concentration failed to exhibit enhancement of radiation sensitivity in H460 cells when added either 24 hour pre- or post-IR treatment (Supplementary Fig. S1A, B). We further extended our pilot observation of combining IR and Abemaciclib (24 hour post-IR treatment) across a panel of lung cancer cell lines with varied genetic background (26) (See Table 1). Abemaciclib (10 µM) when added 24 hour post-IR enhanced radiosensitivity for majority of NSCLC cell lines (Fig. 1A) with DMF ranging between 1.3-1.71. Abemaciclib toxicity and DMF for each cell line is shown in Table1.

Interestingly, cells deficient for functional p53 (H460DNp53, H1299 and H1650) were non- responsive to Abemaciclib + IR combination (Fig. 1B). However, H1975 cells, which does have a mutated p53, was highly sensitive to Abemaciclib incubation (Fig. 1A), and likewise to IR exposure alone (27). H82 cells, lacking functional RB protein, showed no radiosensitization by

Abemaciclib (Fig. 1B). Importantly, the normal mammary epithelial cell line MCF-10A, was not radiosensitized by Abemaciclib with a DMF of 1.03±0.12 (Fig. 1C). All data described to this point apply to exponentially growing cells. Since tumors often have a large fraction of non- proliferating cells, studies combining Abemaciclib and IR exposure was evaluated using quiescent/plateau phase H460 and H460DNp53 cells. Fig. 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

H460DNp53 plateau phase cells did not respond to Abemaciclib chemoradiation (DMF, 1.04 ±

0.01) (Fig. 1D). Collectively, our data indicate Abemaciclib as a novel radiation modifier for

NSCLC cells, independent of mutated EGFR and KRAS, but dependent on a functional RB and possibly on a functional p53 protein status.

Abemaciclib significantly inhibits radiation-induced DNA repair with post-IR treatment

We further investigated the influence of Abemaciclib on IR induced DNA damage repair by assessment of γ-H2AX (at Ser139 ) induction and the time dependent resolution which is indicative of DNA double strand break repair (28). Fig. 2A shows γ-H2AX kinetics following 7.5 Gy and

Abemaciclib treatment in H460 and H1299 cells. Following 7.5 Gy γ-H2AX rapidly increased in

H460 (7.9 fold at 0.5 hour) and H1299 (6.7 fold at 0.5 hours) cells, but resolved to unirradiated control levels (1.1 and 1.2 fold, respectively) 24 hours post-IR. With post-IR Abemaciclib treatment incubation, γ-H2AX remained elevated in H460 (3.92 fold at 24 hour) compared to untreated control (Fig. 2A) indicating inhibition of DNA damage repair. However, H1299 cells treated with Abemaciclib post-IR resulted in similar γ-H2AX repair kinetics to IR alone treated cells over a 24 hour period, which is consistent with lack of Abemaciclib radiosensitization in this cell line (Fig. 2A). To further evaluate the impact of Abemaciclib on DNA damage repair, expression of key DNA repair proteins such as Rad51, phosphorylated Chk2, and ATM following

Abemaciclib or Abemaciclib + IR were observed to be reduced in H460 cells when compared to untreated control (Fig 2B and C). A reduction of phosphorylated ATR (P<0.005, **) and DNA-

PKc (P<0.005, ***) with Abemaciclib and IR combination also suggested a significant DNA-DSB repair inhibition. Importantly, no significant changes in the expression of the above DNA-DSB repair proteins were observed for the p53 deficient cell line, H1299 (Fig. 2D and Supplementary

Fig. S2A). We confirmed the inhibition of RB phosphorylation at Ser780 in H460 (Fig. 2E) and

H1975 cells (Supplementary Fig S2B,) as well as at Ser 811/807 (Supplementary Fig. S2C) in H460 cells after 10µM of Abemaciclib treatment for 24 hours as compared to a 4 hour drug exposure.

Reduced RB phosphorylation post Abemaciclib was followed by a concomitant increase in p21 expression (Fig. 2E and Supplementary Fig S2B) leading to an increased G1 cell cycle arrest (See

Table 1). Abemaciclib incubation also reduced the expression of RB/E2F target proteins such as

TopoIIα (marker for G1/S transition) (Fig. 2E and Supplementary Fig S2B) and showed a significant decrease in p-HH3 (histone H3) expression at Ser 10 (marker for mitosis) as observed by flow cytometry (Supplementary Fig. S2D). Significantly, the G1 block after Abemaciclib incubation was not observed in the RB deficient H82 cell line (Table 1). There was also an accumulation of late apoptotic cells (Annexin V positive + PI positive) after 24 hours of

Abemaciclib and IR treatment in H460, but not in H1299 and H460DNp53 (Supplementary Fig.

S2E) cells. Taken together, our results suggest that inhibition of CDK4/6 activity by Abemaciclib in NSCLC cells causes enhanced G1 arrest and inhibits DNA-DSBs repair pathways leading to enhanced IR sensitization.

Abemaciclib inhibits mTOR signaling in vitro

Another signaling pathway known to influence IR sensitivity is the mTOR pathway that also regulates tumor growth, tumor cell metabolism, and cell stress responses (29,30). Multiple marker proteins of mTOR activation were clearly reduced by Abemaciclib incubation in H460 cells (Fig.

3A). A reduction in expression of p-Akt (Ser 473), p-S6, p-4EBP1, p-p70S6K (Fig. 3A) was observed. Similar observations were made in H1975 cells (data not shown). mTOR also regulates

ATP and total NAD levels (29, 30) as shown in Fig. 3B and C, respectively. ATP and total NAD levels in H460 cells were reduced by 35% and 29% by Abemaciclib and IR incubation compared to IR only, respectively (Fig. 3B and C). Reduction in ATP levels by Abemaciclib is important as chromatin-remodeling complexes, which are ATP-dependent play an important role in all three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non- homologous end-joining (31). Reduced intracellular ATP by Abemaciclib may play a role in inhibition of IR-induced DNA damage repair thereby causing enhanced radiosensitivity.

Collectively, these results suggest that Abemaciclib mediated inhibition of mTOR could possibly lead to reduced intracellular ATP and total NAD levels thereby rendering NSCLC cells vulnerable to enhanced radiosensitization.

Abemaciclib alters NSCLC metabolism by disrupting key nutrient sensing pathways

In recent years, it has been well appreciated that cancer cells have the ability to rewire their metabolism and energy production networks in response to altered microenvironment conditions to support rapid proliferation, invasion, metastasis, and resistance to cancer treatment (32,33). mTOR remains central in regulating tumor metabolism (29,30) . Our study indicated sustained mTOR inhibition by Abemaciclib and Abemaciclib+IR combination (Fig 3A). Hence, we next evaluated the effects of Abemaciclib + IR combination in NSCLC on tumor cell metabolism.

Steady state levels of 116 metabolites in H460 cells using targeted capillary electrophoresis time of flight (CE-TOF) and tandem triple quadruple mass spectrometry (QqQMS) was determined.

Glycolytic intermediates such as fructose 1, 6 bisphosphate were elevated (p<0.005, **) after 24 hour of Abemaciclib treatment, whereas downstream intermediates such as 2-PG (2- phosphoglycerate) and 2-PEP (2-phosphoenylpyruvate) were reduced (p<0.005 ** and p<0.05, *, respectively) suggesting that Abemaciclib altered the activity of glyceraldehyde 6 phosphate dehydrogenase (G6PD). With exception of reduced levels of citrate, most TCA cycle metabolites did not show any significant change after Abemaciclib treatment (Supplementary Fig. S3A).

Interestingly, Abemaciclib caused a significant decrease in steady state levels of both non-essential amino acid (NEAA) and essential amino acids (EAA) (p<0.0001***), suggesting a possible amino acid deficiency (nutrient stress) in H460 cells (Fig 4A and Supplementary Fig. S3B). One of the best-studied ways by which cells maintain their amino acid levels during starvation or nutrient stress in vitro is by elevating general amino acid control (GAAC) pathway (34). Studies have

indicated that glutamine depletion can also trigger the GAAC pathway, by elevating expression of amino acid transporters such as ASCT2, thereby increasing amino acid uptake and elevating intracellular amino acid levels, such as that of Leucine (Leu). Increased Leu has been shown to reactivate mTOR (35,36). Interestingly, in our metabolic analysis, Abemaciclib significantly downregulated levels of Leu (Supplementary Fig. S3B). In addition to changes in steady state levels of glycolytic and TCA cycle metabolites we also observed the kinetics of glucose and glutamine uptake by H460 cells using a YSI-Bioanalyzer. In the presence of Abemaciclib both glucose and glutamine uptake were decreased (Fig. 4B and C). In agreement with the observed decrease in Leu, glutamine and glucose uptake, Abemaciclib treatment altered the surface distribution of both ASCT2 (shown in red, panel D) and glucose transporter (GLUT1, shown in green panel B) (Fig. 4D). Other surface proteins, such as CD98 (also known as 4F2HC or SLC3A2, neutral amino acid transporter) and CD147, a chaperone protein that mediates transport of MCT1 and MCT4 to the plasma membrane, were not affected. Collectively, Abemaciclib treatment reduced ATP levels and restricted access to key metabolic substrates mainly amino acids, glutamine, and glucose possibly leading to nutrient and energy stress. These results indicate that

Abemaciclib exposure leads to sustained mTOR inhibition, limits access to extracellular amino acid pools, and decreases intracellular ATP levels thereby facilitating NSCLC to become sensitive to IR.

Abemaciclib inhibits IR induced vasculogenesis and tumor re-growth in combination with fractionated IR

A pilot H460 xenograft study was conducted combining fractionated IR (three 2 Gy fractions) with daily Abemaciclib (100 mg/kg) treatment through oral gavage. Initially, two daily

Abemaciclib doses (immediately after radiation and 6 hours later) were delivered immediately

after the first IR fraction and the second day; however, this was not well tolerated and subsequently

Abemaciclib was given as daily single dose for the remainder of the week (Wed-Fri). This regimen of Abemaciclib treatment resulted in modest radiosensitization (Supplementary Fig. S4A) and an increased delay in the tumor regrowth kinetics (p=0.02) as compared to radiation only control, followed with rapid regrowth. In an attempt to further enhance tumor regrowth delay we considered the tumor vasculature. Targeting tumor vasculature has long been considered for both radiation and chemoradiation as a means of tumor destruction (37). The novel finding of Brown et al. who demonstrated using a brain tumor xenograft model, that tumor vasculature can recover post-IR by colonization of circulating BMDC (bone marrow derived cells) primarily CD11b+ myeloid cells. This pathway of blood vessel formation, secondary to tumor angiogenesis which is inhibited by IR (38), known as vasculogenesis, restores tumor vasculature facilitating recurrence of tumor following radiation (39,40). They further showed that the stimulus for the influx of

CD11b+ cells into tumors following IR is increased levels of hypoxia (and HIF-1 expression) that drives secretion of the chemokine stromal cell-derived factor-1 (SDF-1). The kinetics of vasculogenesis in the brain tumor model was observed to be as early as two weeks post radiation leading to enhanced tumor re-growth (39). Hence, we hypothesized that should fractionated IR during the first week of treatment initiate vasculogenesis, then an additional week of adjuvant

Abemaciclib treatment might provide additional tumor regrowth delay.

To test this hypothesis, we conducted a second in vivo study where mice with H460 xenografts were exposed to fractionated IR (2 Gy) on Monday, Wednesday and Friday in addition to daily gavage of the Abemaciclib (100 mg/kg) immediately after IR for five days in a week (1st week) followed by an additional five daily treatments of Abemaciclib only without IR (2nd week). In the absence of radiation, Abemaciclib displayed a modest effect on H460 xenograft regrowth by 2

days (p=0.14) versus control untreated tumors (Fig. 5A). However, Abemaciclib in combination with IR showed synergistic delay in tumor regrowth of 8 and 9 days, respectively as compared to

IR only and control tumor (p=0.006 and p=0.001, respectively). Adding a second week of adjuvant

Abemaciclib treatment enhanced tumor delay compared to one week of treatment (Supplemental

Fig. S4A). This study was repeated with similar findings as shown in (Supplementary Fig. S4C).

We next evaluated tumor tissue at the end of the second week of adjuvant treatment to determine if IR induced vasculogenesis occurred and if Abemaciclib inhibited the induction. Reduced immunohistochemical staining of fluorescently labeled CD11b+ monocytes, SDF-1, CD45+ and

F4/80 positive macrophages were observed in week 2 tumors following Abemaciclib and IR +

Abemaciclib combination treatment. In good agreement with Brown et al, week 1 tumor post IR treatment did not show enhanced SDF-1 (40) and CD11b+ BMDC infiltration (data not shown).

Abemaciclib alone inhibited RB phosphorylation and tumor cell proliferation as seen by reduced p-HH3 and Ki-67 staining (Fig. 5B and C). Inhibition of vasculogenesis in week 2 tumors by

Abemaciclib and IR treatment was quantified and shown in Fig 5C. Western analysis of tumor lysates confirmed modest RB inhibition as seen by cyclin A expression in Abemaciclib alone and combination with IR in week 1 tumors (Supplementary Fig. S4B). In good agreement with reduced p-RB expression in week 2 tumor sections (Fig. 5C) we also observed a sustained inhibition of cyclin A expression upon Abemaciclib and Abemaciclib + IR combination in week 2 tumors

(Supplementary Fig. S4B). These results clearly show that inhibition of CDK4/6 pathway leads to inhibition of IR-induced tumor vasculogenesis. Furthermore, in H460 xenografts Abemaciclib treatment led to a sustained inhibition of p-S6 levels indicating reduced mTOR activation

(Supplementary Fig. S4B). IR treatment enhanced expression of HIF-1 at week one, was dramatically inhibited by Abemaciclib (Fig. 5D). Further, Abemaciclib in an adjuvant setting also

inhibited IR induced induction of HIF-1 at week 2, but not completely (Fig. 5D). Abemaciclib inhibited HIF-1 expression independent of IR in untreated tumors (week 2) as tumor growth enhanced the hypoxic fraction (Fig. 5D). The mechanism of how Abemaciclib inhibits induction of HIF-1 in vivo remains to be established and will be a topic of further studies.

Discussion:

With recent development and FDA approval of highly specific CDK4/6 inhibitors (Palbociclib,

Ribociclib and Abemaciclib) for advanced metastatic breast cancer, there has been growing interest in design of multiple clinical trials with these agents in various solid tumors

(https://clinicaltrials.gov/). Successful phase I trial of Abemaciclib in patients with advanced stage

NSCLC and other solid tumors is very encouraging regarding the design of future combination therapy (20). However, recent reports of Abemaciclib failure as monotherapy in Phase III trial for

KRAS mutant NSCLC has been discouraging (http://www.ascopost.com/News/58135). There is nonetheless potential combinatorial therapies where CDK4/6 inhibitors may be beneficial. Pre- clinical studies have indicated that Palbociclib can sensitize KRAS mutant NSCLC cells to MEK inhibitor and EGFR-TKI (22). Ribociclib was shown to sensitize PIK3CA mutant breast cancer to

PI3K inhibitors (41). Pre-clinical studies have shown Palbociclib to modify IR response in breast cancer (42) , glioblastoma (43) and medulloblastoma (44). Our pre-clinical data strongly supports

Abemaciclib, as a potent radiation modifier in NSCLC; whereas, our pilot studies indicates that

Palbociclib and Ribociclib did not yield IR enhancement. This could possibly be tumor-type dependent effect of the drug, drug concentration, and the IR treatment strategy used in our study.

Despite CDK inhibitor successes in managing patients with advanced disease (14) , there are still challenges in the optimization of CDK inhibitors in clinical practice. A major hurdle is lack of predictive biomarkers to screen appropriate patient populations for better therapeutic outcome.

While previous in vitro studies suggested RB, cyclin D, and p16 could predict the response to

Palbociclib (45), results from Phase II/III trials showed no significant correlation between drug response and the expression of p16 (45), Ki67, cyclin D1 amplification (46), PIK3CA or ESR1

(47) mutational status, leaving no established prognostic or predictive biomarkers.

The pre-clinical findings of the current study provide the basis for future clinical trials that are biomarker integrated in NSCLC by combining Abemaciclib with IR. Clonogenic cell survival data across a panel of cell lines harboring genotypic mutations in key driver proteins involved in the progression of NSCLC such as mutation in KRAS, EGFR-TK domain, p53, RB and p16 indicated that Abemaciclib when administered immediately post-IR primarily radiosensitizes cells with functional p53 and RB proteins , but is independent of the mutational status of EGFR, KRAS and p16 protein (Fig. 1 and Table 1). Interestingly, H1975 cells were radiosensitized by Abemaciclib

(Fig. 1A) but have a mutated p53 (48) (Table 1). It has been reported for NSCLC that certain p53 mutations can be non-disruptive leading to a Gain-of-function (GOF) and patients with these mutations have poorer prognosis than patients with disruptive p53 mutations (48). H1975 cells were identified as having a SNP in exon 8 (R273H) which was found to be non-disruptive (48).

The H1975 cells used in this study was also found to have the R273H mutation through whole exon sequencing (data not shown) thus suggesting a possible explanation for our IR sensitization results. Additional research is certainly needed to clarify this observation that in some cases

Abemaciclib can radiosensitize p53 mutated cells.

Radiosensitization of H460 xenografts using fractionated radiation (Fig. 5), combined with

Abemaciclib, indicated that CDK4/6 inhibition may be an excellent target for clinical radiotherapy.

Our data attributes the radiosensitization effect of Abemaciclib to several parallel mechanisms in vitro and in vivo. In vitro, Abemaciclib and IR combination resulted in significant inhibition of IR-

induced DNA damage repair as assessed by γ-H2AX levels and compromised activation of key

DNA damage response proteins to repair DNA (Fig. 2). In the present study, Abemaciclib at 10

µM for 24 hours leads to sustained inhibition of mTOR both in vitro and in vivo (Fig. 3A and

Supplementary Fig. S4B). mTOR inhibition by Abemaciclib further leads to reduced amino acid pools as seen by global metabolic profiling (Fig. 4A). These alterations in the nutrient and energy status of cells possibly increased activated stress responses which lead to cell death via induction of apoptosis in vitro. Clearly, Abemaciclib affects mTOR signaling resulting in reduction in ATP levels and other essential metabolite which may interfere with IR-induced DNA damage repair.

However, the mechanism of mTOR inhibition by Abemaciclib is unclear and requires additional studies.

Most interestingly, our study implicates a unique role of CDK4/6 inhibition in IR-induced vasculogenesis in vivo as a contributor to the enhanced IR response. Inhibition of IR-induced vasculogenesis by Abemaciclib is in good agreement with the findings of Brown et al., who showed that IR to a tumor can induce tumor vasculogenesis leading to recurrence in a GBM xenograft model (39). He further demonstrated that an inhibitor of vasculogenesis (SDF-1/CXCR4 pathway inhibitor, AMD3100) when administered the second week post-IR resulted in enhanced tumor regrowth delay (39) . Phase I/II trial studies evaluating AMD3100 (plerixafor) after radiation therapy and temozolomide in patients with high grade GBM have shown promising local tumor control (49). Our data are consistent with these findings in that SDF-1, while not expressed in the tumor samples the first week of treatment, was enhanced during the second week post-IR

(Fig. 5B and C). Abemaciclib treatment during the second week inhibited SDF-1 induction (Fig.

5B and C), thus inhibiting vasculogenesis. In addition to inhibiting IR mediated vasculogenesis

(Fig. 5) CDK4/6 inhibition also exerted sustained mTOR inhibition and of HIF-1 in vivo. CDK4/6

treatment also prevented tumor cell entry to mitosis as shown by reduced p-HH3 and Ki-67 staining (Fig. 5C). These findings are crucial from the point of success of radiotherapy because both enhanced tumor hypoxia (induction of HIF-1) and mTOR activation are well-documented mechanisms for altering radiosensitivity in tumors (24,50). Collectively, our results suggest that there are multiple mechanisms of Abemaciclib radiosensitization including inhibition of DNA damage repair, mTOR signaling, and IR induced vasculogenesis.

In summary, the results of the present study suggest that Abemaciclib and IR combination would be a reasonable way to manage NSCLC patients in early and advance disease settings with implications on minimizing IR induced tumor recurrence. Our findings clearly warrant consideration of this novel chemoradiation combination in clinical trials.

Acknowledgements

We thank to Lim Langston and Poonam Mannan (NCI Core Imaging Facility, NIH) for technical assistance on confocal microscopy image capture and analysis.

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Table 1: Summary of Abemaciclib effect on NSCLC cell lines Toxicity, Cell Cycle, and Radiation Sensitivity

Radiation Mutational Status Cell Cycle Toxicity Effect Cell Lines Histology % % % EGFR RB1 KRAS TP53 p16 PTEN DMF(SD) PE (%) G1 S G2 78 4 13 H460 LCC DEL? WT MUT WT DEL WT 1.71(0.15) 22 (66) 79 11 10 H460 DN p53 LCC WT WT MUT DN DEL WT 1.14(0.02) 50 (61) 90 8 2 A549 NSCLC WT WT MUT WT DEL WT 1.43(0.02) 60 (72) 77 13 9 H1299 NSCLC WT WT WT DEL WT WT 1.01(0.01) 85 (55) 69 10 21 H1975 NSCLC MUT WT WT MUT WT WT 1.63(0.02) 54 (49) 82 15 3 H820 NSCLC MUT WT MUT MUT WT WT 1.3(0.51) 66 (68) 64 16 20 H1650 NSCLC MUT WT WT MUT DEL 1.0(0.03) 56 -/- (51) 51 20 29 H82 SCLC WT WT WT WT WT 1.02(0.03) 77 -/- (49) Normal 87 5 8 MCF10A Amp WT WT MUT DEL WT 1.03(0.12) 59 Breast (49) 94 2 4 H460 plateau LCC WT WT MUT WT DEL WT 1.35(0.15) 33 (92) H460 DN p53 89 7 4 LCC WT WT MUT DN DEL WT 1.04(0.01) 49 plateau (79)

Values in parenthesis are the % G1 of untreated cells. (-/-) represent null status for gene, Amp: Gene Amplification, MUT: Mutated gene, DN: Dominant Negative gene, WT: wild type gene. MCF710A are immortalized Normal Breast Epithelial cells. Mutational Status of most cell lines determined based on Blanco et al., (26) and Molina-Vila MA et al., (48). LCC: Large Cell Carcinoma; NSCLC: Non-Small Cell Lung Cancer; SCLC: Small Cell Lung Cancer. DMF: dose modifying factor, PE: plating efficiency. SD: Standard Deviation. Toxicity was based on a 10 uM Abemaciclib exposure for 24 hr.

Figure Legends

Figure 1. Radiation survival curves for lung cancer cell lines treated with Abemaciclib. (A-

D). All cells were treated with Abemaciclib (10 µM, dashed curve with closed circles) or with

DMSO control (0.1%, solid curve with open circles) for 24 hours immediately post irradiation and clonogenic assay was 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.

Figure 2. Effect of Abemaciclib on radiation induced DNA damage repair and CDK4/6-RB pathway in NSCLC cells. (A) Western blot analysis of phosphorylated γ-H2AX in isolated histones from H460 and H1299 cell lines as a function of time following treatment with

Abemaciclib (10 µM) 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 (F.C) 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 + IR (7.5 Gy) combination. (C-D) Densitometry analysis of three independent experiment in H460 cells (C) and H1299 cells (D) post-IR and Abemaciclib treatment. The expression of p-ATR, p-ATM and p-Chk2 were normalized based on their corresponding total protein expression. The error bar represents mean ± SEM (Standard Error of

Mean) from three independent experiment. 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.

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,1:1000) post Abemaciclib treatment in the presence or absence of IR. (B-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 is represented as pmoles of

ATP normalized to total cell number. Error bar represents mean ± SEM (Standard Error of Mean) and statistical significance is indicated as ** p<0.01 and *** p<0.001.

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

(non-essential 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 is 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 (µg) of protein in the sample. Experiments were done in triplicates and error bar represent mean ± SD (standard deviation). * p<0.01, ** p<0.001 and *** p<0.0001. (D) Immunofluorescence analysis of H460 cells treated with DMSO or Abemaciclib for

24 hour showing expression of Glut-1 (glucose transporter, shown in green panel A and B, respectively) and ASCT2 (amino acid transporter, shown in red panel C and D, respectively).

DAPI was used to stain nucleus (shown in blue). Images were taken at 60X using Zeiss 780 confocal microscope. Scale bar is 10 µm.

Figure 5. Effect of Abemaciclib and fractionated radiation on tumor re-growth and radiation induced vasculogenesis. (A and B) H460 xenograft showing enhanced tumor regrowth delay by daily administration of 100mg/kg body weight of Abemaciclib through oral gavage for 5 days

immediately after 2 Gyx3 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 800mm3. The statistical significance is derived from 8 animals per group. (B). Confocal microscopy showing expression of Ki-67, p-

HH3,p-RB and infiltration of BMDC (bone marrow derived cells) such as CD45, CD11b+ monocytes and F4/80+ 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 100x. Scale bar is 10µm. (C) Mean florescence intensity (MFI) of immunopositive cells of indicated proteins were measured for approximately eight random fields of the immunostained section using Image J. The fold expression was calculated by normalizing the MFI to that of MFI of DMSO treated tumor section. (D) Immunoblot analysis showing expression of HIF-1α in H460 xenograft for week 1 and week 2 of Abemaciclib administration post fractionated radiation.

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Abemaciclib, a Selective CDK4/6 Inhibitor Enhances the Radiosensitivity of Non-Small Cell Lung Cancer in vitro and in vivo

Sarwat naz, Anastasia L. Sowers, Rajani Choudhuri, et al.

Clin Cancer Res Published OnlineFirst May 1, 2018.

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