Mechanisms of Efficacy of the FGFR1–3 Inhibitor AZD4547 in Pediatric Solid Tumor Models

Nikki Phanhthilath 1 & Sara Hakim 1 & Catherine Su 1 & Andrea Liu 1 & Divya Subramonian 1 & Jacqueline Lesperance 1 &
Peter E. Zage 1,2

Received: 3 February 2020 /Accepted: 7 April 2020
# Springer Science+Business Media, LLC, part of Springer Nature 2020

Summary
Children with aggressive pediatric solid tumors have poor outcomes and novel treatments are needed. Pediatric solid tumors demonstrate aberrant expression and activity of the fibroblast growth factor receptor (FGFR) family, suggesting FGFR inhibitors may be effective therapeutic agents. AZD4547 is a multikinase inhibitor of the FGFR1–3 kinases, and we hypothesized that AZD4547 would be effective in pediatric solid tumor preclinical models. We evaluated the effects of AZD4547 on neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma cells alone and in combination with STAT3 inhibition. Continuous live cell imaging was used to measure induction of apoptosis and effects on migration. Receptor inhibition and intracellular signaling were examined by western blotting. AZD4547 treatment resulted in decreased cell confluence, increased apoptosis and reduced cell migration in all tested cell lines. AZD4547 treatment led to decreased phosphorylation of signaling proteins involved in cell survival and apoptotic pathways and increased phosphorylation of STAT3, and treatment of cell lines with AZD4547 combined with STAT3 inhibition demonstrated increased efficacy. Sensitivity to AZD4547 appears to be mediated by effects on the Ras/MAPK and JAK/STAT pathways, and AZD4547 represents a potential novel therapeutic agent for children with solid tumors.

Keywords Neuroblastoma . Rhabdomyosarcoma . Ewing sarcoma . AZD4547 . FGFR . STAT3

Abbreviations IGF-1R insulin-like growth factor-1 receptor

FGFR fibroblast growth factor receptor
DMSO dimethyl sulfoxide
RIPA radioimmunoprecipitation assay
MAPK mitogen-activated protein kinase
VEGFR2 vascular endothelial growth factor-2

ERK
ATCC
extracellular signal-regulated kinase American Type Culture Collection
Introduction

PLCγ phospholipase-C gamma (PLCγ)
STAT3 signal transducer and activator of transcription 3
Children with aggressive solid tumors such as neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma have poor outcomes

despite intensive multimodal treatment that frequently in-

Nikki Phanhthilath and Sara Hakim contributed equally to this work. Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10637-020-00933-2) contains supplementary material, which is available to authorized users.

* Peter E. Zage [email protected]
cludes multiple cycles of myelosuppressive chemotherapy, surgical tumor resection, and focal radiation therapy, with tumors that are resistant to treatment and frequently recur. Refractory and recurrent pediatric solid tumors respond poorly to salvage therapy [1–5], and survivors experience significant side effects and complications as a result of this therapy. Currently, the causes of these solid tumors are mostly un-

1
Department of Pediatrics, Division of Hematology-Oncology, University of California San Diego, 3855 Health Sciences Dr., MC 0815, La Jolla, CA 92093, USA
known, and the pathways and signals involved in their growth and spread are not well understood. Children with these ag- gressive solid tumors desperately need new types of treatment,

2 Peckham Center for Cancer and Blood Disorders, Rady Children’s Hospital, San Diego, CA 92123, USA
and newer therapies directed against targets and pathways important for cancer cell survival and resistance to treatment

are likely to improve the chances of both survival and cure and reduce the frequency and severity of long-term side effects.
Aberrant growth factor receptor (GFR) expression and re- ceptor kinase signaling are common features of malignant transformation and tumorigenesis, and these kinases therefore represent targets for a number of novel therapies. Fibroblast growth factors (FGFs) and their receptors (FGFR1–4) take part in key biological processes such as embryonic develop- ment tissue repair, and angiogenesis, and aberrant FGFR sig- naling has been implicated in several malignancies [6, 7]. FGFR activation plays an important role in tumorigenesis through stimulation of proliferation and migration and inhibi- tion of cell death [8–13]. Furthermore, FGFR inhibition has been shown to be effective in a variety of preclinical cancer models, including pediatric cancers [14–24].
AZD4547 is a novel, orally active inhibitor of the FGFR family of receptor tyrosine kinases, with IC50 values for FGFR1, FGFR2, FGFR3, and FGFR4 of 0.2 nM, 2.5 nM, 1.8 nM, and 165 nM, respectively, in cell-free assays [25]. AZD4547 has been proven effective in preclinical models of numerous adult and pediatric cancers [25–37]. However, the efficacy of AZD4547 against most pediatric solid tumor types has not been well established. Based on the potential roles for these targets in pediatric solid tumor pathogenesis, we hypoth- esized that inhibition of FGFR family members using AZD4547 would be effective against pediatric solid tumors.

Materials and Methods

Cells and Culture Conditions The neuroblastoma cell lines used in this study have been previously described [38–40]
and were generously provided by Susan Cohn (The University of Chicago Children’s Hospital, Chicago, IL), ac- quired from the Children’s Oncology Group (www.cccells. org), or were purchased from the American Type Culture Collection (ATCC; Rockville, MD). Rhabdomyosarcoma cell lines (Rh30, RD, Rh41, Rh5) were generously provided by Javed Khan (National Cancer Institute, Frederick, MD), Jason Yustein (Baylor College of Medicine, Houston, TX), acquired from the Children’s Oncology Group or were purchased from the ATCC. Ewing sarcoma cell lines (A673, RDES, SK-ES-1, TC32, TC71) were generously provided by Jason Yustein, Jing Yang (University of California San Diego, San Diego, CA), or were acquired from the Children’s Oncology Group. Cells were grown in RPMI-1640 medium (SK-N-AS, SK-N-SH, SK-N-BE(2), IMR-32, RDES, SK-ES- 1, Rh5, Rh30, Rh41) or Dulbecco’s Modified Eagle Medium (TC32, TC71, A673, RD) and supplemented with L-gluta- mine, 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA) and antibiotics and antimycotics at 37 °C in 5% CO2. All cell lines were authenticated by deoxyribonucle- ic acid (DNA) profiling prior to use.

Therapeutic Agents N-[5-[2-(3, 5-Dimethoxyphenyl) ethyl]- 2H-pyrazol-3yl]-4-(3, 5-dimethylpiperazin-1-yl) benzamide (AZD4547, AstraZeneca, Cambridge, UK) was generously pro- vided by AstraZeneca. A 10 mM stock solution was prepared in DMSO and stored at -80 °C. AZD4547 was diluted in culture media to desired concentrations immediately before use. Stattic was purchased from Selleck Chemicals (Houston, TX).

Cell Confluence Assays Cells were plated in 96-well plates at seeding densities between 10,000–25,000 cells/well. After 24 h, AZD4547, Stattic, or the combination of AZD4547 with Stattic was added to each well. Phase contrast images were taken every 6 h at 10X magnification for 72 h using IncuCyte Zoom™ imaging system (Essen Bioscience, Ann Arbor, MI). Cell growth curves were generated from percent cell conflu- ence acquired from the IncuCyte Zoom™ analyzer. Replicates of at least three wells were used for each experimental condi- tion, and assays were performed at least three independent times. IC50 values were derived using best-fit trendlines and calculated using the appropriate curve-fit equations.

Caspase 3/7 Apoptosis Assays Cells were plated in 96-well plates at seeding densities between 10,000–25,000 cells/well. After 24 h, cells were treated with AZD4547 and IncuCyte Caspase 3/7 Green Reagent™ (Essen Bioscience) was added. Phase contrast and fluorescence images were taken every 6 h at 10X magnification for 72 h using the IncuCyte Zoom™. Average green object counts (representing individual apoptotic cells) were obtained and fold apoptosis was calculated and nor- malized to the control. Replicates of at least three wells were used for each experimental condition.

Fig. 1 FGFR Family Member Expression in Pediatric Solid Tumor Cell Lines. Total cell lysates from neuroblastoma (NB), rhabdomyosarcoma (RMS), and Ewing sarcoma (EWS) cell lines were analyzed by Western blot for expression of FGFR1–4. GAPDH was used as a loading control.

Fig. 2 AZD4547 Reduces Pediatric Solid Tumor Cell Confluence. (A) Cell confluence was analyzed following 72 h of AZD4547 treatment using continuous live cell imaging for neuroblastoma (NB), rhabdomyosarcoma (RMS), and Ewing sarcoma (EWS) cell lines. (B)

Photographs were taken after 72 h of treatment to document cell appearance. (C) IC50 values were calculated and cells are listed from lowest to highest IC50 values for each tumor type.

Wound Healing Assay Cells were plated in ImageLock 96- well plates (Essen Bioscience) at a seeding density of 45,000–125,000 cells/well and allowed to adhere overnight. 24 h after plating, uniform horizontal scratches in each well were generated by a WoundMaker (Essen Bioscience), followed by AZD4547 treatment. Scratch wound images were taken every 6 h at a 10X magnification for 48 h using the IncuCyte Zoom™. Wound width was calculated and normal- ized to the control wells. Replicates of at least three wells were used for each experimental condition.

Western Blots Cells were plated in 6-well plates or 10 cm dishes at approximately 70–80% confluency and were allowed to adhere overnight. For single agent studies, cells were treated with AZD4547 for 1 h followed with 20 ng/ml of bFGF (R&D Systems, Minneapolis, MN) for 3 h. For com- bination studies, cells were treated with AZD4547 and Stattic for 4 h. Cells were washed with cold PBS buffer and lysed using RIPA buffer supplemented with Protease inhibitor and phosphatase inhibitor (Life Technologies, Carlsbad, CA). Protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, San Diego, CA). Equal amounts of protein were loaded onto 4–12% Bis-Tris gels

(Invitrogen, Carlsbad, CA) with MOPS SDS Running Buffer and transferred to PVDF membranes. Membranes were blocked by 5% BSA/1X TBS + 0.1% Tween 20 and incubated overnight with primary antibodies to total STAT3, phospho- STAT3, total ERK, phospho-ERK, total S6, phospho- S6, total Akt, phospho-Akt (Ser473), phospho-FGFR, total FGFR1, FGFR2, FGFR3, and FGFR4 (all from Cell Signaling, Danvers, MA), and β-actin (Sigma- Aldrich, St. Louis, MO). Anti-rabbit or anti-mouse HRP-conjugated secondary antibody (1:5000, Sigma- Aldrich, St. Louis, MO) incubation was performed at room temperature for 1 h and membranes were devel- o p e d u s i n g S u p e r S i g n a l ™ W e s t P i c o P l u s Chemiluminescent Substrate (Thermo Fisher Scientific).

Results

Neuroblastoma, Rhabdomyosarcoma, and Ewing Sarcoma Cell Lines Demonstrate Mixed Patterns of FGFR Expression To determine whether pediatric solid tumor cells might be sensitive to FGFR inhibition, total cell lysates from neuroblasto- ma, rhabdomyosarcoma, and Ewing sarcoma lines were

Fig. 3 AZD4547 Induces Pediatric Solid Tumor Cell Apoptosis. (A) Images of neuroblastoma (SK-N-BE(2), SK-N-AS), rhabdomyosarcoma (Rh30, RD), and Ewing sarcoma (TC-71, A673) cell lines were taken following

treatment with AZD4547 for 72 h with green dots representing individual apoptotic cells. (B) Fold change in apoptosis was analyzed by normalizing the average green object count/mm2 to the control (*p < 0.05).

analyzed by Western blot for expression of the FGFR1–4 recep- tors. All tested solid tumor cell lines demonstrated expression of

at least three FGFR family members (Fig. 1), suggesting that FGFR inhibition may be effective against these cells.

Fig. 4 AZD4547 Inhibits Pediatric Solid Tumor Cell Migration. (A) Neuroblastoma (SK-N-BE(2), SK-N-AS), rhabdomyosarcoma (Rh30, RD), and Ewing sarcoma (TC-71, A673) cells were plated and a scratch wound was generated. Images were obtained following the initial scratch

wound and after 48 h of treatment with AZD4547 or vehicle (veh) alone, with cells migrating into the scratch wound demarcated in purple. (B) Wound width was calculated at regular intervals and changes in wound width were plotted over time and normalized to control (*p < 0.05).

AZD4547 Treatment Reduces Pediatric Solid Tumor Cell Confluence and Induces Apoptosis To evaluate the effects of AZD4547 on the viability and growth of pediatric solid tumor cell lines, neuroblastoma, rhabdomyosarcoma, and Ewing sar- coma cell lines were monitored using continuous live cell imaging during treatment with AZD4547. Cell lines were ex- posed to increasing doses of AZD4547 for 72 h and cell con- fluence was calculated. The majority of cell lines demonstrat- ed similar sensitivity to AZD4547, with calculated IC50 values between 2.56 and 8.65 μM (Fig. 2, Supplemental Fig. 1). However, the SK-N-AS neuroblastoma cells and the RD rhabdomyosarcoma cells proved to be more resistant to the drug, with IC50 values over 20 μM (Fig. 2).
To determine the mechanism of reduced cell confluence, AZD4547 treated cells were analyzed for apoptosis. Induction of apoptosis was detected at 2.5 μM AZD4547 in Rh30 cells, 5 μM AZD4547 in SK-N-AS, and RD cells, and at 10 μM AZD4547 in all other tested cell lines (Fig. 3, Supplemental Fig. 2).

AZD4547 Inhibits Pediatric Solid Tumor Cell Migration FGFR signaling has been shown to be critical for regulation of cell migration and invasion, hallmarks of cancer metastasis. To investigate the ability of AZD4547 to inhibit cell migration, pediatric solid tumor cells were subjected to a scratch wound assay. A uniform scratch wound generated in cells grown as a confluent monolayer and treated with sublethal doses of AZD4547 was monitored using continuous live cell imaging over 48 h. All cell lines shown to be sensitive to AZD4547 demonstrated reduced rates of wound closure with AZD4547 treatment (Fig. 4, Supplemental Fig. 3), suggesting that AZD4547 has additional potential anticancer effects.

AZD4547 Inhibits Multiple Intracellular Signaling Pathways We next investigated the ability of AZD4547 to inhibit kinase targets and critical intracellular signaling pathways. Despite minimal effects on FGFR family member phos- phorylation, AZD4547 inhibited ERK, Akt, and S6 phos- phorylation in all tested cell lines (Fig. 5, Supplemental

Fig. 5 AZD4547 Inhibits Multiple Intracellular Signaling Pathways. (A) Neuroblastoma (SK-N-BE(2), SK-N-AS), rhabdomyosarcoma (Rh30, RD), and Ewing sarcoma (TC-71, A673) cells were treated with AZD4547 prior to stimulation with bFGF (20 ng/mL). Western blots were performed to determine effects on levels of total and phosphorylated ERK1/2, AKT, S6, and STAT3. (B) Neuroblastoma,

rhabdomyosarcoma, and Ewing sarcoma cells were treated as above and Western blots were performed for total and phosphorylated FGFR. β-actin was used as a loading control in both experiments. Relative band intensity levels in Western blots for phosphorylated ERK1/2, AKT, S6, and STAT3 are listed as percentages of untreated cells.

Fig. 4). Sensitive cell lines demonstrated increased levels of phosphorylated ERK after ligand stimulation and inhi- bition of ERK phosphorylation after treatment, while other cell lines demonstrated less robust inhibition of ERK phos- phorylation. No apparent association was detected between cell line sensitivity and inhibition of Akt phosphorylation. Interestingly, sensitive cell lines demonstrated a dramatic increase in phosphorylation of STAT3 after AZD4547 treatment, while the SK-N-AS and RD cell lines demon- strated decreased or unchanged levels of phospho-STAT3, suggesting STAT3 phosphorylation represents a potential biomarker for AZD4547 response and resistance.

The Combination of AZD4547 and Stattic is Effective against Pediatric Solid Tumor Cell Lines To evaluate whether inhi- bition of STAT3 might enhance responses of pediatric solid tumor cell lines to AZD4547, neuroblastoma, rhab- domyosarcoma, and Ewing sarcoma cell lines were mon- itored using continuous live cell imaging during treatment with AZD4547 combined with the STAT3 inhibitor Stattic. The combination resulted in significantly reduced cell confluence compared to either drug alone in both AZD4547 sensitive and resistant cell lines (Fig. 6). AZD4547 combined with Stattic also reduced STAT3 phosphorylation in AZD4547 sensitive cell lines but not in the SK-N-AS and RD cell lines (Fig. 6), suggesting that other signaling pathways may contribute to the AZD4547 resistance in these cell lines.

Discussion

Children with aggressive and relapsed solid tumors such as neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma have poor outcomes despite intensive treatment [1–5], and children with these aggressive solid tumors desperately need new ther- apies. Therapies directed against targets and pathways impor- tant for cancer cell survival and resistance to treatment are likely to improve the chances of both survival and cure and reduce the frequency and severity of long-term side effects. We therefore evaluated the efficacy and mechanisms of action of AZD4547, a FGFR1–3 kinase inhibitor [25], in neuroblas- toma, rhabdomyosarcoma, and Ewing sarcoma cell lines.
Aberrant FGFR expression and activity have been identi- fied in numerous cancer types [7, 41], and altered expression and activity are the result of a variety of genetic defects, in- cluding gene amplification, chromosomal translocation, re- ceptor overexpression and activating point mutations. Increased FGFR expression and oncogenic activation results in ligand-independent intracellular signaling through a variety of pathways, including the RAS-MAPK, phosphoinositide-3 kinase, phospholipase-C gamma (PLCγ) and signal transduc- er and activator of transcription 3 (STAT3) pathways [42–44]
and subsequent increased angiogenesis along with increased tumor cell proliferation, survival and chemoresistance [45].
FGFR inhibition has been shown to be effective in a variety of preclinical cancer models, including pediatric rhabdoid tu- mors, gliomas, ependymomas, and rhabdomyosarcomas

Fig. 6 AZD4547 Combined with STAT3 Inhibition Reduces Pediatric Solid Tumor Cell Confluence and STAT3 Signaling. (A) Neuroblastoma (SK-N-BE(2), SK-N-AS), rhabdomyosarcoma (Rh30, RD), and Ewing sarcoma (TC-71, A673) cells were treated with AZD4547 in combination with Stattic for 72 h and cell confluence was calculated using continuous

live cell imaging (**P < 0.01 *P < 0.05). (B) Neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma cell lines were treated with AZD4547 and increasing concentrations of Stattic, and cell lysates were analyzed by Western blots for total and phosphorylated STAT3. β-actin was used as a loading control.

[14–24]. However, many of these kinase inhibitors inhibit a number of other kinase targets, and the efficacy of more spe- cific FGFR inhibitors against most types of pediatric solid tumors has not been well established. AZD4547 is a novel, specific inhibitor of the FGFR1, FGFR2, and FGFR3 kinases [25] that has previously been proven effective in preclinical models of myeloma, breast cancer, gastric cancer, endometrial cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, soft tissue sarcoma, and prostate cancer [25–37]. Furthermore, AZD4547 treatment has been well tol- erated by patients in early phase clinical trials, with no dose limiting toxicities observed and primarily gastrointestinal and dermatologic side effects, including altered taste, stomatitis, dry mouth, and diarrhea. Pharmacokinetic analyses demon- strated that the peak drug levels in adult patients reached mi- cromolar concentrations [46], within the range of our in vitro IC50 values and suggesting that effective drug levels may be attainable in children as well, although the safety and tolera- bility of AZD4547 have not been established in children. However, the challenge in selecting appropriate biomarkers for response to AZD4547 and the presence of multiple inde- pendent resistance mechanisms has likely contributed to the lack of significant responses in clinical trials to date in adults with relapsed solid tumors [46–48], suggesting that a better understanding of the genetic and molecular profiles of tumors may allow for identification of patients most likely to respond.
Expression of FGFR family members has previously been identified in pediatric solid tumors, including neuroblastoma [49, 50], rhabdomyosarcoma [20, 51, 52], osteosarcoma [53], and pediatric brain tumors [54–56]. Our results demonstrate similar FGFR expression patterns in neuroblastoma and rhab- domyosarcoma cell lines and further explore the FGFR ex- pression patterns in Ewing sarcoma cell lines. The pediatric solid tumor cell lines tested in these studies demonstrated a range of FGFR family member expression, and these expres- sion patterns did not correlate with responses to AZD4547. Furthermore, AZD4547 treatment did not result in dramatic inhibition of FGFR phosphorylation, despite significant inhi- bition of downstream intracellular signaling pathways, sug- gesting that either AZD4547 is acting on other previously undescribed targets or that only minimal FGFR inhibition is necessary to reduce intracellular signaling critical for cellular migration and survival.
While AZD4547 is a potent inhibitor of the FGFR1–3 re- ceptor tyrosine kinases, at higher concentrations AZD4547 is capable of inhibiting other receptor kinases, including the vas- cular endothelial growth factor receptor -2 (VEGFR2; KDR) and the insulin-like growth factor-1 receptor (IGF-1R) with cell-free IC50 values of 24 nM and 581 nM, respectively [25]. Prior studies have shown that VEGF was capable of bind- ing to a neuroblastoma cell line, with increased cell growth in response to exogenous VEGF [57]. However, while pediatric solid tumor cells have been shown to express VEGF ligands,

more recent studies have shown that the expression of the VEGFR2 receptor is only found at low levels in a small per- centage of pediatric solid tumor cell lines [58, 59]. Recent studies have demonstrated responses of children with Ewing sarcoma to VEGFR2 inhibition with the multikinase inhibitor cabozantinib [60], although prior studies had demonstrated that VEGFR2 inhibitors had little in vitro activity but significant activity in in vivo models [61], suggesting an anti-angiogenic effect rather than a direct effect on tumor cells. Furthermore, treatment of mice with neuroblastoma and Ewing sarcoma xe- nograft tumors with an antibody to mouse VEGFR2 to specif- ically target tumor vasculature was shown to be effective [59], further suggesting that VEGFR2 inhibition is unlikely to be responsible for significant effects in cell lines tested in vitro.
Expression of the IGF1R gene has been found in pediatric solid tumor cell lines and tumor samples [62–64]. Furthermore, signaling through the IGF-1R has been shown to be active in pediatric solid tumor models, specifically in models of Ewings sarcoma, and inhibition of IGF-1R was shown to be effective against pediatric solid tumors, with Ewing sarcoma being the most sensitive and neuroblastoma and rhabdomyosarcoma demonstrating intermediate sensitiv- ity [65–67]. Alternative strategies targeting IGF-1R were also effective against mouse xenograft models of Ewing sarcoma [68]. Therefore, the inhibition of intracellular signaling by AZD4547 in our experiments may also be due in part to IGF-1R inhibition as well as FGFR1–3 inhibition, or may be due to inhibition of other, unidentified kinase targets, and further studies are clearly needed to elucidate the mecha- nism(s) of AZD4547 activity in different tumor types.
Our results demonstrated that the majority of tested neuro- blastoma, rhabdomyosarcoma, and Ewing sarcoma cell lines were sensitive to AZD4547, with AZD4547 treatment induc- ing apoptosis, reducing cell migration, and leading to inhibi- tion of critical intracellular signaling pathways. Our results further show that pediatric solid tumor cell lines sensitive to AZD4547 demonstrate increased STAT3 phosphorylation in response to AZD4547 treatment, and the combination of AZD4547 with STAT3 inhibition is more effective in these cell lines than either alone. STAT3 is constitutively active in a number of cancers and plays a crucial role as a regulator of apoptosis, cell proliferation, and inflammatory responses [69, 70], making it a common target for anti-cancer therapies. Previous studies have also identified roles for the PI3K [71]
and RAS-MAPK pathways [72] in addition to STAT3 [73] in the resistance of cancer cells to AZD4547, consistent with our results and suggesting that combinations of critical pathway inhibitors with AZD4547 are more likely to be effective.
In summary, we have demonstrated that the novel FGFR kinase inhibitor AZD4547 is effective against pediatric solid tumor cell lines. AZD4547 treatment of neuroblastoma, rhab- domyosarcoma, and Ewing sarcoma cell lines results in inhi- bition of critical intracellular signaling pathways, resulting in

reduced migration and induction of apoptosis. Although the specific mechanisms of AZD4547 in indivudual tumor types remain to be elucidated, the demonstrated efficacy of AZD4547 alone and in combination with STAT3 inhibition in three different pediatric solid tumor types suggests that further preclinical testing is warranted.

Acknowledgements This study was supported by AstraZeneca who pro- vided study drug (to PEZ).

Code Availability Not applicable.

Author contributions NP, SH, AL, DS, and JL carried out experiments for the study, and NP, SH, CS, AL, and PEZ compiled the results, gen- erated the figures for the paper, and prepared the manuscript. PEZ con- ceived the study, designed the experiments, directed the project and helped edit and submit the manuscript. All authors read and approved the final manuscript.

Funding information The work was supported by institutional start-up funds to the principal investigator (PEZ) by the Department of Pediatrics at the University of California San Diego.

Data Availability All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).

Compliance with ethical standards

Conflict of interest This study was supported by AstraZeneca who pro- vided study drug (to PEZ). All other authors declare that they have no conflict of interest.

Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent For this type of study, formal consent is not required.

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