Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-Like ALL Keith C.S

Published OnlineFirst August 14, 2020; DOI: 10.1158/1541-7786.MCR-19-1098

MOLECULAR CANCER RESEARCH | CANCER GENES AND NETWORKS

Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL Keith C.S. Sia1, Ling Zhong2, Chelsea Mayoh1, Murray D. Norris1,3, Michelle Haber1, Glenn M. Marshall1,4, Mark J. Raftery2, and Richard B. Lock1,3

ABSTRACT ◥ Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) is combination cytotoxicity assays using the tyrosine kinase inhi- characterized by aberrant activation of signaling pathways and bitors BMS-754807 and ponatinib that target IGF1R and FGFR1, high risk of relapse. Approximately 50% of Ph-like ALL cases respectively, revealed strong synergy against both cell line and overexpress cytokine receptor-like factor 2 (CRLF2) associated patient-derived xenograft (PDX) models of CRLF2r Ph-like ALL. with gene rearrangement. Activated by its ligand thymic stromal Further analyses also indicated off-target effects of ponatinib in lymphopoietin (TSLP), CRLF2 signaling is critical for the devel- the synergy, and novel association of the Ras-associated protein- opment, proliferation, and survival of normal lymphocytes. To 1 (Rap1) signaling pathway with TSLP signaling in CRLF2r Ph- examine activation of tyrosine kinases regulated by TSLP/CRLF2, like ALL. When tested in vivo, the BMS-754807/ponatinib com- phosphotyrosine (P-Tyr) profiling coupled with stable isotope bination exerted minimal efficacy against 2 Ph-like ALL PDXs, labeling of amino acids in cell culture (SILAC) was conducted associated with low achievable plasma drug concentrations. using two CRLF2-rearranged (CRLF2r) Ph-like ALL cell lines Although this study identified potential new targets in CRLF2r stimulated with TSLP. As a result, increased P-Tyr was detected Ph-like ALL, it also highlights that in vivo validation of syner- in previously reported TSLP-activated tyrosine kinases and sub- gistic drug interactions is essential. strates, including JAK1, JAK2, STAT5, and ERK1/2. Interestingly, TSLP also increased P-Tyr of insulin growth factor 1 receptor Implication: Quantitative phosphotyrosine profiling identified (IGF1R) and fibroblast growth factor receptor 1 (FGFR1), both of potential therapeutic targets for high-risk CRLF2-rearranged Ph- which can be targeted with small-molecule inhibitors. Fixed-ratio like ALL.

Introduction Functionally, CRLF2 heterodimerizes with the a subunit of the IL7 receptor (IL7Ra) to form a receptor for the ligand thymic The application of genomic profiling to acute lymphoblastic stromal lymphopoietin (TSLP), which is heavily implicated in the leukemia (ALL) has led to the identification of recurrent genomic activation of immune cells and allergy response (7, 8). Since its alterations that can be used in risk stratification and treatment implication in the etiology of Ph-like ALL, several studies have adaptation (1). Philadelphia chromosome (Ph)-like (or BCR–ABL1- sought to elucidate the CRLF2 downstream signaling network using like) ALL is a high-risk ALL subtype comprising 15% of childhood quantitative phosphoproteomic approaches and genetically modi- and 25% of adult ALL (2) and is identified through its unique gene- fied murine cell line models (9, 10). These studies established the expression profile that resembles Ph-positive ALL, in the absence of activation of several downstream pathways by CRLF2, including the the defining BCR–ABL1 gene fusion (3, 4). Further characterization JAK–STAT, MAPK, and PI3K–AKT signaling pathways. of Ph-like ALL revealed activating mutations in tyrosine kinases At present, pharmacologic inhibition of CRLF2 is not feasible due to (TK) such as JAK1 and JAK2, thus providing a rationale for the the lack of small-molecule inhibitors directly targeting the receptor testing of TK inhibitors (TKI) in this disease (5). complex. As such, a number of studies have sought to target signaling Notably, gene alterations resulting in overexpression of cytokine pathways downstream of CRLF2, such as JAK2, MEK, mTOR, PI3K, receptor-like factor 2 (CRLF2) are found in approximately 50% of and BCL-2, with varying degrees of success (11–14). Of these studies, Ph-like ALL cases, and are associated with poor outcome (6). the combination of ruxolitinib (JAK1/2 inhibitor) and gedatolisib (mTOR/PI3K inhibitor) achieved the best therapeutic outcome in animal models of Ph-like ALL (13). On the other hand, the modest 1 Children's Cancer Institute, School of Women's and Children's Health, UNSW efficacy observed in other studies reflected the redundant and com- 2 Sydney, Sydney, Australia. Bioanalytical Mass Spectrometry Facility, Mark pensatory nature of oncogenic kinase signaling as well as the challenges Wainwright Analytical Centre, UNSW Sydney, Sydney, Australia. 3UNSW Centre for Childhood Cancer Research, UNSW Sydney, Sydney, Australia. 4Kids Cancer in targeting Ph-like ALL using TKIs. Centre, Sydney Children's Hospital, Randwick, Australia. In this study, we stimulated two CRLF2-overexpressing Ph-like ALL cell lines with TSLP, followed by quantitative phosphotyrosine Note: Supplementary data for this article are available at Molecular Cancer fi Research Online (http://mcr.aacrjournals.org/). (P-Tyr) pro ling to identify TSLP/CRLF2-activating TKs. In contrast to previous studies, we used the MHH-CALL-4 and MUTZ-5 Corresponding Author: Richard B. Lock, Children's Cancer Institute, School of Women's and Children's Health, UNSW Sydney, Sydney, NSW 2052, Australia. cell lines that were derived from Ph-like ALL patients with Phone: 612-9385-2513; Fax: 612-9662-6584; E-mail: [email protected] CRLF2 gene rearrangements and activating JAK2 mutations (15), with the aim of identifying clinically relevant targetable pathways. Mol Cancer Res 2020;XX:XX–XX Through this approach, we identified a total of 52 tyrosine- doi: 10.1158/1541-7786.MCR-19-1098 phosphorylated proteins that were upregulated by TSLP, including 2020 American Association for Cancer Research. targetable TKs and signaling pathways that have not been

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previously implicated in TSLP/CRLF2 signaling. Based on these SILAC labeling, P-Tyr peptide enrichment, and mass results, we rationally combined the insulin growth factor 1 receptor spectrometry analysis (IGF1R) inhibitor BMS-754807 and the fibroblast growth factor MHH-CALL-4 and MUTZ-5 cells were cultured in heavy RPMI- fi L 13 L receptor 1 (FGFR1) inhibitor ponatinib to evaluate their ef cacy 1640 media containing -Lysine:2HCl ( C6) and -Arginine:HCl in vitro in vivo 13 15 L and against CRLF2-rearranged (CRLF2r) ALL ( C6; N4; Cambridge Isotope Laboratories), 20 mg/mL -proline patient-derived xenograft (PDX) and cell line models. (Sigma-Aldrich), and supplemented with 20% dialyzed fetal bovine The BMS-754807/ponatinib combination demonstrated potent cell serum (FBS; Sigma-Aldrich). Cells cultured in heavy media were killing effects in vitro against a panel of CRLF2r ALL PDXs, with stimulated with 20 ng/mL TSLP (Thermo Fisher) before cell lysis. limited efficacy observed in CRLF2-wild-type (WT) PDXs. Further- The following steps in protein extraction, peptide purification, immu- more, RNA-sequencing (RNA-seq) analysis of ex vivo–treated noaffinity enrichment of P-Tyr peptides, and liquid chromatography– CRLF2r Ph-ALL PDX cells verified inhibition of candidate pathways tandem mass spectrometry (LC-MS/MS) analysis have been described identified from the quantitative P-Tyr profiling experiments. When in detail elsewhere (20). evaluated in vivo against two CRLF2r Ph-like ALL PDXs, the combination resulted in a significant, but modest, delay in leukemia In vitro cell culture and cytotoxicity assays progression. Subsequent pharmacodynamic and pharmacokinetic Cell lines were cultured in RPMI-1640 media supplemented with studies also revealed insufficient target inhibition when compared either 10% (NALM-6) or 20% (MHH-CALL-4, MUTZ-5) FBS. PDX with in vitro findings, and that the desired drug exposure was not cells were retrieved from liquid nitrogen storage and thawed in a 37 C achieved in vivo. Overall, this study highlights the myriad of TKs and water bath before use. PDX cells were cultured in QBSF-60 media their associated signaling pathways activated by TSLP/CRLF2 in supplemented with 20 ng/mL FMS-like tyrosine kinase 3 ligand CRLF2r Ph-like ALL, their inhibition as potential treatments for (FLT3L). Cell concentration and viability were determined using the Ph-like ALL, but that in vivo validation of combination drug Trypan Blue exclusion assay. Single-agent and combination cytotox- effects is essential. icity assays were carried out as previously described (12), and combination effect was determined based on the Bliss model by Bliss Additivity (BA) deviation values (21). Confidence Intervals were Materials and Methods calculated from the mean BA deviation values at an alpha of 0.05 to Culturing of immortalized cell lines determine synergy (BA > 0) or antagonism (BA < 0). Cell viability was MHH-CALL-4 and MUTZ-5 cells were purchased from determined using Alamar Blue reagent and the Victor X3 plate reader the German Collection of Microorganisms and Cell Cultures (PerkinElmer; at excitation 560 nm, emission 590 nm). Apoptosis GmbH (DSMZ) and authenticated by CellBank Australia (July assays were conducted by staining cells with Annexin V and 7-AAD fi followed by flow cytometry assessment of the proportion of Annexin 2018) through short tandem repeat pro ling. NALM-6 cells were requested from the internal Cell Bank of Children's Cancer V /7-AAD cells. Institute (Sydney, Australia), which stock was authenticated by RNA isolation, purification, and analyses CellBank Australia (August 2018). Cell lines were kept in culture fi fornolongerthan3months,andMycoplasma testing was con- Total RNA was extracted and puri ed using TRIzol (Life Technol- fi ducted every 6 months using the MycoAlert Mycoplasma Detection ogies) and RNeasy kit (Qiagen). Puri ed RNA was converted to Kit (Lonza). cDNA using the Moloney-murine leukemia virus (M-MLV) reverse transcriptase kit (Life Technologies). TaqMan Gene-Expression Mutation profiles of ALL patient-derived xenografts Assay primer sets (Thermo Fisher) were used to measure expression IGF1R FGFR1 PDGFa CRLF2r and Ph-like ALL PDXs were established using biopsies of (Hs00609566_m1), (Hs00241111_m1), c-MYC from patients enrolled in the Children's Oncology Group (COG) (Hs00234994_m1), and (Hs00153408_m1) via real-time quan- P9906 clinical trial and molecularly characterized previously (12). titative reverse transcriptase-polymerase chain reaction (qRT-PCR). EF1a CRLF2-WT B-ALL PDXs have been previously characterized (16). The expression of was used as an endogenous control. Real-time Procedures by which continuousPDXlineswereestablished qRT-PCR was carried out using the ABI 7900HT Fast Real-Time PCR using immune-deficient nonobese diabetic/severe combined immu- System (Thermo Fisher). fi scid node ciency (NOD/SCID (NOD.CB17-Prkdc /SzJ) or NOD/ Electroporation and siRNA gene silencing / scid tm1Wjl SCID/IL-2 receptor gamma (NOD.Cg-Prkdc IL2rg / Cells were electroporated with the Gene Pulser Xcell (Bio-Rad) SzJAusb, NSG) mice, as well as monitoring of leukemia engraftment using a preoptimized square wave protocol (300 V for 15 ms, 2 pulses at fi and puri cation of human mononuclear cells from engrafted 5 seconds apart). The candidate genes IGF1R and FGFR1 were knocked – spleens, have been described in detail elsewhere (16 18). Key down using the Stealth siRNA oligo sets (HSS179797 and HSS142012, genomic alterations of each PDX are detailed in Supplementary respectively; Thermo Fisher) at a final concentration of 100 nmol/L. Table S1. mRNA expression of candidate genes was determined by real-time qPCR, and cell viability was assessed by Alamar Blue assay, at 48 and Protein expression analysis 72 hours following electroporation, respectively. Procedures for the preparation of whole-cell protein lysates, determination of protein concentration, and immunoblotting RNA-sequencing have been described in detail elsewhere (19). All primary antibodies RNA samples were sent to Novogene for cDNA library construction were purchased from Cell Signaling Technology, with the exception and sequencing (paired-end, 150 bp) using the Illumina HiSeq of CRLF2 (Thermo Fisher) and actin (Sigma-Aldrich). Both anti- platform. The resulting RNA-seq reads were mapped to the human mouse and -rabbit secondary antibodies were supplied by Sigma- genome assembly (build hg38) using STAR (version 2.5) with Aldrich. Antibody details can be provided upon request. quantMode parameter set to TranscriptomeSAM for alignments

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translated into transcript coordinates. The transcriptomic align- and quantitation of the analytes was performed in multiple reaction ments were run through RSEM (version 1.2.31) command rsem- monitoring mode. calculate-expression to calculate gene and isoform expression. Differentially expressed genes were identified with R statistical software using the edgeR package through Bioconductor. Results Quantitative P-Tyr profiling of TSLP-stimulated MHH-CALL-4 In vivo drug efficacy testing and MUTZ-5 cell lines All in vivo experiments were carried out with approval from the We first validated if TSLP could further induce phosphorylation of Animal Care and Ethics Committee of UNSW Sydney (Sydney, candidate proteins in both MHH-CALL-4 and MUTZ-5 cells, to Australia). NSG mice were inoculated with 5 106 PDX cells ensure changes in phosphorylation can be detected in the subsequent each in groups of 9 per treatment arm. Engraftment was monitored quantitative P-Tyr profiling experiments. JAK2, STAT1, STAT3, þ weekly by flow-cytometric enumeration of the human CD45 pop- STAT5, AKT, and ERK1/2 are key components of the JAK–STAT, þ ulation in the mouse peripheral blood (PB, %huCD45 ) as previously MAPK, and PI3K–AKT signaling pathways, all of which are associated described (16, 18). Mice were randomized and treatments commenced with CRLF2 signaling and therefore expected to be constitutively þ when the median huCD45 reached ≥ 1% in each group. BMS-754807 active (9). Shown in Fig. 1A, markedly increased phosphorylation of and ponatinib were purchased from Synkinase. BMS-754807 (in 80% these candidate proteins was observed in both MHH-CALL-4 and Tween-20/20% water; v/v) was administered twice a day 6 hours apart MUTZ-5 cells upon stimulation with TSLP for 30 minutes, in contrast at 25 mg/kg via oral gavage. Ponatinib (in 25 mmol/L citrate buffer) to the findings in NALM-6 (CRLF2-WT) cells. The results were was administered once a day at 15 mg/kg via oral gavage. consistent between serum-starved cells and those cultured in the þ The %huCD45 was monitored weekly throughout treatment as a presence of 20% serum. Furthermore, the majority of these proteins surrogate measure of leukemia progression, and an event was con- examined were not constitutively active in NALM-6 cells, in contrast to þ sidered to occur when the %huCD45 reached 25%. Event-free both CRLF2-overexpressing cell lines. In concordance with the immu- survival (EFS) was determined from the initiation of treatment, and noblot results, positive effects of TSLP on the metabolic activity and drug efficacy was assessed by calculating the difference in median EFS viability of MHH-CALL-4 and MUTZ-5 cells were confirmed using between the vehicle (C) and treatment (T) groups (leukemia growth the Alamar Blue assay, which was not observed in NALM-6 (Supple- delay; T–C) and visualized using Kaplan–Meier survival curves (22). mentary Fig. S1A). In addition, the effects of TSLP on cell viability and The Gehan–Breslow–Wilcoxon test was used to compare significant signaling activation were attenuated using anti-TSLP antibodies (Sup- differences in survival curves between groups. plementary Fig. S1B and S1C). These results confirmed the validity of using TSLP to stimulate CRLF2-mediated signaling pathways in Pharmacokinetic study of BMS-754807 and ponatinib in NSG MHH-CALL-4 and MUTZ-5 cell lines. mice Next, quantitative P-Tyr profiling was carried out to identify Na€ve NSG mice were treated with single agents BMS-754807 targetable TKs that are activated by TSLP/CRLF2 in both and ponatinib or their combination at doses and schedules identical MHH-CALL-4 and MUTZ-5 cells. Briefly, the candidate cell lines to the efficacy study. At 1, 6, and 24 hours after treatment adminis- were cultured in either normal (Light; L) or SILAC (Heavy; H) media tration, plasma samples were collected from 3 mice per treatment for a minimum of five cell cycles, followed by stimulation with TSLP for group. LC-MS analysis was performed on a Shimadzu 8060 triple 30 minutes in only the SILAC-labeled cells. Then, equal parts of L and quadrupole instrument coupled with a Shimadzu Nexera X2 UHPLC. H protein extracts were combined for each cell line, followed by MS analysis was conducted in positive mode electrospray ionization, immunoaffinity enrichment of Tyr-phosphorylated peptides and

Figure 1. Activation of signaling pathways by TSLP in MHH-CALL-4 and MUTZ-5 cells. A, MHH-CALL-4, MUTZ-5, and NALM-6 cell lines were cultured in both serum-depleted and 20% serum- supplemented media conditions. TSLP was added to the culture media at a ﬁnal concentration of 20 ng/mL for 30 minutes. Both phosphorylated and total protein expression of candidate proteins were investigated via immunoblotting. Expression of actin was used as a loading control. B, Visual- ization of functional protein–protein interactions between TSLP/CRLF2- activating proteins using STRING. Enrichment of signaling pathways was carried out against the KEGG database and implicated proteins were color-coded accordingly. C4, MHH- CALL-4; M5, MUTZ-5; N6, NALM-6.

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Table 1. List of TSLP-upregulated proteins in MHH-CALL-4 and MUTZ-5 cells as determined by a minimum of 1.5 normalized H/L ratio and potential small-molecule inhibitors.

Normalized Small-molecule Normalized Small-molecule Protein H/L ratio inhibitor Protein H/L ratio inhibitor

UBE2F 13.2580 FLT3 5.7726 Sunitinib INSR 13.1020 BMS-754807 STX7 5.6285 NRP1 11.6890 EG00229 SPRY2 5.6204 CD46 11.5877 C10orf90 5.5074 SMAGP 11.4100 APLP2 5.2926 ELFN2 10.8143 SLC12A2 5.2508 FLT1 10.5868 Linifanib SIGIRR 4.999 PLD3 9.3170 AFG3L2 4.7877 LAPTM4A 9.0257 FGFR1 4.7021 Ponatinib INSR; IGF1R 9.0135 BMS-754807 ITSN2 4.4388 MAPK1 8.9771 SCH772984 STAT5A; STAT5B 4.1166 Ruxolitinib MAPK3 8.9768 SCH772984 TYK2 3.8889 TGOLN2 8.9262 CALM1 3.5209 PTPN11 8.7643 AS1949490 ERBB3 3.3904 Sapitinib NPDC1 8.6193 ACTB 3.2245 SLC12A6 8.0193 F2R 3.0778 SIGLEC15 7.9046 CNBP 2.873 LRIG1 7.6639 MAPK14 2.7426 Skepinone-L SV2A 7.3107 EPHB4 2.6542 IL2RG 7.0646 CRIP1 2.4183 CRIM1 6.9074 ELFN1 2.0497 GAB1 6.7564 JAK1 2.0304 Ruxolitinib NEDD9 6.7380 F11R 1.9671 APP 6.6212 JAK2 1.9016 Ruxolitinib CMTM6 6.0754 SLC25A5 1.8267 BST2 6.0255 ITGB1 1.6674

LC-MS/MS analyses as previously described (20). Summarized in more, each protein was color-coded according to their involvement in Supplementary Fig. S2A, a total of 329 phospho-sites (including serine, the top five KEGG-enriched signaling pathways (Fig. 1B; Supplemen- threonine, and tyrosine) were identified with localization probabilities tary Table S2). ranging from 1 to 0.24, and 259 sites were considered as class I (>75% Established CRLF2-activated signaling pathways such as the PI3K– localization probability; Supplementary Fig. S2B), of which 85% were AKT, RAS, and JAK–STAT were among the enriched pathways P-Tyr sites. Importantly, 96% of all L P-Tyr sites were paired with (Fig. 1B; Supplementary Table S2). Interestingly, the Ras-associated their H counterparts (Supplementary Fig. S2C), and hence allowing protein-1 (Rap1) signaling pathway, which has not been associated quantitative comparison of peptide intensity between experimental with CRLF2 signaling previously, was the top-ranked enriched path- conditions (23). way. From this analysis, IGF1R, FGFR1, MAPK1 (ERK1), and MAPK3 Using the MaxQuant software, the measured intensity of both H (ERK2) were the kinases involved in the most enriched signaling and L P-Tyr sites was normalized and the H/L ratios were determined. pathways (n ¼ 4). In addition, we subjected the list of TSLP-activated Using a minimum cutoff of 1.5 to the normalized ratios, a total of 52 proteins to GO analyses (Biological Process and Molecular Function). proteins that were at least 1.5-fold upregulated upon TSLP stimulation The top 5 ranked pathways from both analyses and their associated were identified among the MHH-CALL-4 and MUTZ-5 cells (Table 1), proteins are listed in Supplementary Table S2. The GO analysis results with 42 downregulated proteins identified at the same threshold. exemplified the aberrant kinase activation in Ph-like ALL, where Where available, small-molecule inhibitors are listed next to their mostly protein phosphorylation and kinase-related pathways were targets. As anticipated, proteins such as JAK1, JAK2, STAT5, MAPK1 enriched, for instance peptidyl-tyrosine phosphorylation, transmem- (ERK1), and MAPK3 (ERK2) were upregulated by TSLP (9, 10). Of brane receptor protein tyrosine kinase signaling pathway, and protein interest, targetable kinases such as INSR/IGF1R and FGFR1, which tyrosine kinase activity. have not been implicated in CRLF2 signaling previously, were also found to be upregulated upon TSLP stimulation. In vitro cytotoxicity testing of BMS-754807 and ponatinib in To visualize signaling networks activated by TSLP/CRLF2 in our Ph-like ALL study, the 52 TSLP-activating proteins were submitted to STRING for Based on the above findings, we proceeded with dual targeting of functional protein–protein interaction analysis (24), followed by IGF1R and FGFR1 using the TKIs BMS-754807 and ponatinib, pathway analysis through the KEGG database (25). The protein– respectively. The single-agent activity of the inhibitors was assessed protein interaction network presented in Fig. 1B was built based on in CRLF2r cell line and PDX models. As shown in Supplementary high confidence (0.9) and previously reported evidence, where an Fig. S3A and S3B, both TKIs demonstrated moderate efficacy as single intricate network was mapped around 21 protein nodes and two agents against the cell lines. BMS-754807 achieved IC50 values of 2.6 independent interactions involving two pairs of proteins. Further- and 1.2 mmol/L in MHH-CALL-4 and MUTZ-5 cell lines, respectively,

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where treatment with ponatinib resulted in slightly lower IC50 parison with the single agents and their expected additive effects values of 1.4 mmol/L (MHH-CALL-4) and 0.3 mmol/L (MUTZ-5). (Fig. 2A; Supplementary Table S3). This synergy was further elabo- Using the same assay, we evaluated the potency of these TKIs rated using three CRLF2r PDX models (Fig. 2B; Supplementary against a panel of six CRLF2r B-ALL PDXs, five of which are Ph-like Table S3). In PAMDRM, the combination was able to achieve ALL (with the exception of PALNTB). Overall, the panel of PDXs < 20% cell viability relative to controls even at the lowest concentra- responded to both agents similarly, with median IC50 values of 0.5 tions tested (125 nmol/L of each inhibitor). Moreover, to determine if mmol/L for BMS-754807 (Supplementary Fig. S3C) and 1.0 mmol/L the synergy was specific to CRLF2r PDXs, the combination was also for ponatinib (Supplementary Fig. S3D). The PDX PALLSD tested against three B-ALL PDXs without CRLF2 alteration. The BMS- ¼ appeared to be slightly more sensitive to BMS-754807 (IC50 754807/ponatinib combination was antagonistic in ALL-2 and addi- 52.6 nmol/L) than the rest of the PDXs tested. Furthermore, tive in ALL-7 (Fig. 2C; Supplementary Table S3). However, the immunoblotting was carried out to confirm the dephosphorylation combination was synergistic against ALL-19, which may be due to of IGF1R and FGFR1 following 1 hour of drug treatment (1 mmol/L; the presence of the NUP214–ABL1 translocation in this PDX, because Supplementary Fig. S3E). ABL1 is a target of ponatinib. Importantly, however, the rationally Next, we conducted fixed-ratio combination testing of BMS-754807 designed combination of BMS-754807 and ponatinib exhibited prom- and ponatinib to determine their synergistic effects in CRLF2r ALL. ising synergy in all of the CRLF2r cell lines and PDXs tested, thereby The combination treatment was synergistic in both MHH-CALL-4 prompting further investigation into its potential mechanism of action and MUTZ-5 cells, as indicated by the greater cytotoxicity in com- and in vivo efficacy.

Figure 2. Fixed-ratio combination effect of BMS-754807 and ponatinib against candidate cell lines and PDXs. The single agents were combined in ﬁxed ratios and tested against (A) CRLF2r cell lines MHH-CALL-4 and MUTZ-5; (B) CRLF2r ALL PDXs PALLSD, PALNTB, and PAMDRM; and (C) CRLF2-WT ALL PDXs ALL-2, ALL-7, and ALL-19. Target cells were exposed to each of the single agents and their respective combinations for 72 hours. Cell viability was assessed using Alamar Blue cytotoxicity assay, and each data point represents the mean SEM of three independent experiments.

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In addition to using pharmacologic inhibitors, we sought to inves- against preclinical models of Ph-like ALL (13). On the other hand, the tigate if silencing of the candidate genes by siRNA would result in Rap1 signaling pathway may represent a novel signaling pathway to be similar effects on the two cell lines. IGF1R expression was reduced by exploited in the treatment of CRLF2r Ph-like ALL. 60% to 65% in both cell lines, while FGFR1 expression was reduced Next, we sought to confirm differential expression of two candidate by approximately 75% (Supplementary Fig. S4A). Although IGF1R genes, PDGFa and MYC, using orthologous methodology (real-time knockdown resulted in 35% to 40% loss of viability in both cell lines qRT-PCR). PDGFa was one of the genes that was significantly down- (Supplementary Fig. S4B), FGFR1 knockdown caused only a 20% loss regulated only by the combination treatment and is involved in both of viability in MHH-CALL-4 cells and no notable loss of viability in the PI3K–AKT and Rap1 signaling pathways. Furthermore, its recep- MUTZ-5 cells. Moreover, the combined effects of knockdown of both tor, PDGFRa, was previously implicated in the resistance mechanism genes reflected the effects of IGF1R knockdown alone. These results toward BMS-754807 (26). Expression of PDGFa appeared to be suggest that the potent combination effect of BMS-754807 and pona- slightly upregulated by BMS-754807 in the RNA-seq analysis. How- tinib could be attributed to off-target effects of ponatinib. ever, it was downregulated by ponatinib and to a greater extent by the combination treatment (Fig. 3D). Validation by real-time qRT-PCR Investigation of potential underlying mechanisms behind the revealed a similar trend in the expression of PDGFa. synergy between BMS-754807 and ponatinib observed in vitro The MYC protooncogene encodes a transcription factor activated using RNA-sequencing analysis by JAK/STAT, MAPK, and PI3K/AKT signaling pathways in the In an effort to elucidate mechanisms underlying the prominent context of hematopoiesis (27). Furthermore, c-MYC was previously synergy observed in vitro, RNA-seq was conducted on PAMDRM reported to be a downstream target of IL7R, and their combined PDX cells. Time course apoptosis assays were performed to determine inhibition was implicated in the sensitivity of CRLF2r Ph-like ALL the appropriate drug exposure time, where the PDX cells were treated toward the BET bromodomain inhibitor, JQ1 (28). As shown with either single agent or their combination for 48 hours. The effects in Fig. 3E, the regulation of MYC expression was almost identical on cell viability were not apparent until at least 16 hours of drug between the RNA-seq and real-time qRT-PCR results, where MYC was exposure (Fig. 3A). In order to capture the upstream changes in gene markedly downregulated by both BMS-754807 and the BMS-754807/ expression leading to the synergistic cell killing, RNA samples were ponatinib combination. prepared from three biological replicates of PAMDRM cells exposed to Overall, results from the RNA-seq analysis revealed that BMS- the designated drug treatments for 12 hours. 754807 had a greater impact than ponatinib on gene-expression In the RNA-seq analysis we first constructed a multidimensional changes in PAMDRM cells. When combined with ponatinib, it scaling (MDS) plot to visualize the relationship between samples. As resulted in a significantly higher number of differentially expressed shown in Fig. 3B, each treatment exerted unique changes to the genes. Moreover, the RNA-seq results also implicated the involvement transcriptome of PAMDRM cells, resulting in clustering of samples of the Rap1 signaling pathway in CRLF2r Ph-like ALL. into individual treatment groups. From the MDS plot, it was also apparent that the combination treatment caused the largest changes in In vivo efficacy testing of BMS-754807 and ponatinib in Ph-like the transcriptome in relation to the controls. Subsequently, we iden- ALL tified differentially expressed genes from each treatment group against The combination treatment of BMS-754807 and ponatinib was control using the criteria of false discovery rate (FDR) < 0.05 and next evaluated in vivo against two CRLF2r Ph-like ALL PDXs. Prior to FC >|2|, as visualized using volcano plots (Fig. 3C). As expected, the the efficacy study, we conducted tolerability testing in na€ve NSG mice combination treatment resulted in the highest number of differentially to establish appropriate doses of the combination. BMS-754807 expressed genes (776; Supplementary Table S4), followed by the single (25 mg/kg) was tested in combination with varied doses of ponatinib agents BMS-754807 (452; Supplementary Table S5) and ponatinib (25–6.25 mg/kg). The median and individual mouse % weight change (129; Supplementary Table S6). for each treatment group over time are shown in Supplementary To infer signaling pathways affected by each treatment, lists of Fig. S5. Toxicity leading to >15% weight loss was observed in two differentially expressed genes from each treatment were submitted for mice from the highest treatment group, and one experienced >20% pathway analysis against the KEGG database, and the results are weight loss. The remaining groups experienced a median weight loss of summarized in Supplementary Table S7. The combination treatment <10%. As an additional precaution for when animals are engrafted had the greatest number of enriched signaling pathways at 19, while with leukemia, the drug doses selected for the efficacy study were ponatinib only resulted in a single enriched pathway. Several signaling 25 mg/kg BMS-754807 and 15 mg/kg ponatinib. pathways enriched in the BMS-754807 single-agent treatment group Both single agents were unable to significantly delay the progression were also implicated in the combination treatment group, albeit with of the PDXs PALLSD or PAMDRM relative to vehicle control–treated more gene counts, likely due to the added effect of ponatinib. For mice, with the exception of ponatinib against PAMDRM (T–C ¼ example, by BMS-754807 treatment 18 differentially expressed genes 4.1 days, P ¼ 0.011; Fig. 4A and B; Table 2). The BMS-754807/ were enriched in the cytokine–cytokine receptor interaction and 9 in ponatinib combination resulted in significant, but modest, delay in the the JAK–STAT signaling pathway, while there were 27 and 15 impli- progression of both PDXs. We also extracted RNA from PAMDRM cated genes respectively as a result of the combination treatment. cells derived from the spleens and bone marrows of mice 6 hours after Two of the signaling pathways previously enriched in the P-Tyr treatment to assess the expression of candidate genes previously profiling analysis, Rap1 and PI3K–AKT signaling pathways, were interrogated in the RNA-seq analysis as indicators of drug activity. affected by only the combination treatment in the RNA-seq analysis. As shown in Fig. 4C, the expression of PDGFa was not inhibited by any The exclusive impacts on these proposed TSLP/CRLF2-induced sig- treatments, in contrast to the in vitro findings. Instead, both single naling pathways by the combination treatment could potentially agents elevated the expression of PDGFa, which was further upregu- contribute to the prominent synergy observed in vitro. Especially with lated in the combination treatment. Furthermore, the expression of the PI3K–AKT signaling pathway, its combined inhibition with the MYC, which was dramatically downregulated by BMS-754807 and the JAK/STAT pathway was recently demonstrated to be highly effective combination in vitro, was also unaffected in vivo.

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Figure 3. Investigation of potential underlying mechanisms behind the synergy observed between BMS-754807 and ponatinib using RNA-sequencing analysis. A, Prior to conducting RNA-seq, apoptosis assays were carried out to determine the appropriate drug exposure length in PAMDRM PDX cells. Cell viability was determined using flow cytometry by measuring the proportion of Annexin V/7-AAD cells as compared with controls. Error bars represent mean range of at least two independent experiments. From the subsequent RNA-seq of ex vivo–treated PAMDRM PDX cells (n ¼ 3; B), the relationship between samples was inferred through MDS analysis based on the 500 most heterogeneous genes between each sample pair. C, Defined by FC > |2| and FDR < 0.05, the differentially expressed genes caused by each treatment group were visualized in green in the volcano plots. D and E, Expression of candidate genes was quantified using real-time qRT-PCR, normalized to that of the internal control, EF-1a, and expressed as fold change over control. The results are plotted alongside values from the RNA-seq analysis for comparison. Error bars represent mean SEM of three biological replicates. CTRL, control; BMS, BMS-754807; PON, ponatinib; COMB, combination of BMS-754807 and ponatinib; FC, fold change; FDR, false discovery rate.

We suspected that sustained drug exposure was required for the was still evident at 48 hours drug exposures, albeit to a lesser extent synergistic interactions observed between BMS-754807 and ponatinib when compared with that of 72 hours (Fig. 2B). At 24-hour drug in vitro, which may not be achieved in vivo. Therefore, we repeated the exposures, the combination only appeared to be synergistic at higher in vitro fixed-ratio combination cytotoxicity assays with shorter drug drug concentrations (Supplementary Fig. S6B). exposures to determine if the combination remained synergistic. As To confirm whether the plasma levels of each drug achieved in the shown in Supplementary Fig. S6A, the synergy had diminished in in vivo efficacy experiment were lower than those required to exert PALLSD cells at the shorter time points of 24 and 48 hours of drug cytotoxic synergy in vitro, we conducted a pharmacokinetic study to exposure. In PAMDRM cells (Supplementary Fig. S6B), the synergy measure the plasma concentrations of BMS-754807 and ponatinib at

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Figure 4. In vivo efﬁcacy evaluation of BMS- 75807, ponatinib, and their combination against CRLF2r Ph-like ALL PDXs in NSG mice. A and B, Engraft- ment of leukemia as measured by the %huCD45þ cells over time is shown on the left, while the Kaplan–Meier survival curves are shown on the right for (A) PALLSD and (B) PAMDRM. Gray shad- ing indicates the treatment period. C, Total RNA was extracted from PAMDRM cells treated in vivo and iso- lated from both bone marrows and spleen of engrafted mice. The expression levels of PDGFa and MYC tran- scripts were measured using real- time qRT-PCR and normalized against that of the control gene, EF-1a. Error bars represent mean range from two biological replicates. BM, bone marrow; SPL, spleen.

specific time points following drug administration. As presented in presented in Supplementary Fig. S6, the minimum drug concentra- Supplementary Fig. S7 and Table S8, both drugs achieved maximum tions required to achieve synergy in PAMDRM at 24 hours drug concentrations within 6 hours of administration, followed by steady exposures were between 0.25 and 0.5 mmol/L, while the concentrations decreases to less than 30 nmol/L of either single agent present in the of BMS-754807 and ponatinib detected in mouse plasma at 24 hours plasma at 24 hours after treatment. Putting into context with the results were approximately 4 and 28 nmol/L, respectively, when administered in combination (Supplementary Table S8). These results also ruled out the possibility of negative interaction between the two drugs Table 2. In vivo efficacy of BMS-754807 and ponatinib against Ph- evident from the similar pharmacokinetic profiles between single like ALL PDXs. agents (Supplementary Fig. S7A) and combination (Supplementary Significance (P value) Fig. S7B) treatments. Together, these results suggest that the drug in vitro Median EFS T–C BMS- exposures similar to those required for drug synergy were not PALLSD (d) (d) Vehicle 754807 Ponatinib achieved in vivo, resulting in the limited efficacy against both PDXs.

Vehicle 5.5 BMS-754807 5.7 0.2 ns Discussion Ponatinib 5.8 0.3 ns ns Combination 6.1 0.6 0.0153 ns ns Activating mutations of cytokine receptors and TKs are recurrently found in Ph-like ALL (5, 29). Although CRLF2r occurs in approxi- fi Significance (P value) mately 50% of Ph-like ALL cases, there are currently no speci c small- Median EFS T–C BMS- molecule inhibitors available for its targeting. This study aimed to PAMDRM (d) (d) Vehicle 754807 Ponatinib identify targetable TKs activated by TSLP/CRLF2 for novel targeted treatments of CRLF2r Ph-like ALL. To date, two phosphoproteomic Vehicle 11.2 analyses of TSLP signaling in the context of malignancy have been BMS-754807 11 0.2 ns Ponatinib 15.3 4.1 0.0111 ns carried out using human CRLF2 overexpressed in murine cell line fi Combination 14.3 3.1 0.0023 0.0047 ns models, both of which have contributed signi cantly to understanding oncogenic CRLF2 signaling (9, 10). The current study distinguishes

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itself in the use of human CRLF2r cell lines immortalized from Ph-like malignancies, Rap1 signaling has been shown to promote migration ALL patients. and invasiveness of B-cell lymphoma and T-cell ALL (35–37). Because Based on STRING analysis of proteins activated by TSLP/CRLF2 of its role in cell adhesion and interactions, Rap1 signaling is likely to be in our study, we targeted IGF1R and FGFR1 because they were important in vivo, by facilitating the interaction between leukemia cells involved in four of five enriched signaling pathways, and both also and the bone marrow microenvironment. represent novel therapeutic targets in CRLF2r Ph-like ALL. Aber- Finally, due to the myriad of downstream signaling pathways rant signaling of IGF1R and FGFR1 have been previously impli- activated by TSLP/CRLF2, direct targeting of CRLF2 is promising cated in cancer, although mainly in solid tumors (30, 31). The for the treatment of CRLF2r Ph-like ALL. A number of studies have combination of BMS-754807 and ponatinib yielded synergistic cell investigated the potential of antibody-based targeting options as killing effects against two Ph-like ALL cell lines, and greater effects well as antibody-conjugated nanoparticles in the context of both against CRLF2r Ph-like ALL PDXs. Importantly, the combination leukemia and allergy (38–40). Furthermore, promising results were was less effective when tested against ALL PDXs without CRLF2 also observed in CRLF2 targeting chimeric antigen receptor (CAR)- alterations. Out of the three PDXs tested, the combination only T cells (41). As Ph-like ALL patients suffer a higher risk of relapse, exerted synergy in vitro in ALL-19, which was likely due to the which remains one of the leading causes of mortality in children, a NUP214–ABL1 translocation present in the PDX, as ABL1 is a curative treatment targeting Ph-like ALL is highly likely to further principal target of ponatinib. siRNA knockdown experiments indi- improve the overall outcome of childhood ALL. cated that the potent synergy of the combination could be attributed to off-target effects of ponatinib. Disclosure of Potential Conflicts of Interest Despite the modest in vivo efficacy of the drug combination, which No potential conflicts of interest were disclosed. was likely due to an inability to achieve the required plasma concentrations of both drugs, certain findings from this study merit further Authors’ Contributions investigation. For instance, the potential of IGF1R as a therapeutic K.C.S. Sia: Conceptualization, formal analysis, funding acquisition, validation, – – target in CRLF2r Ph-like ALL is also supported by our previous study, investigation, visualization, methodology, writing original draft, writing review, and editing. L. Zhong: Formal analysis and methodology. C. Mayoh: Software, formal where elevated phosphorylation of INSR/IGF1R was found among Ph- analysis, visualization, and methodology. M.D. Norris: Resources, writing–review, like ALL PDXs, in comparison with those derived from other ALL and editing. M. Haber: Resources, writing–review, and editing. G.M. Marshall: subtypes (20). Together with increased phosphorylation observed Resources, writing–review, and editing. M.J. Raftery: Resources and supervision. through TSLP stimulation in this study, these data indicate further R.B. Lock: Conceptualization, resources, supervision, funding acquisition, project testing of INSR/IGF1R targeting despite the lack of efficacy observed administration, writing–review, and editing. with BMS-754807. For instance, instead of TKIs, monoclonal antibodies targeting INSR/IGF1R could be used, which possess more Acknowledgments favorable pharmacokinetic properties. In fact, a number of monoclo- This research was supported by the National Health and Medical Research Council of Australia (NHMRC Fellowships APP1059804 and APP1157871 to R.B. Lock, nal antibodies targeting the INSR/IGF1R signaling have been tested in NHMRC Program Grant 1091261), the Cancer Council NSW (Program Grant PG16- clinical trials mainly for solid tumors (32). 01) and the Steven Walter Children's Cancer Foundation. K.C.S. Sia was supported by From our study and others, it is apparent that TSLP/CRLF2 a UNSW Postgraduate Scholarship and top-up scholarships from Cancer Therapeu- activates a wide range of downstream signaling pathways, including tics CRC and Kids Cancer Alliance. the JAK–STAT, MAPK, and PI3K pathways. In this study, we also fi The costs of publication of this article were defrayed in part by the payment of page identi ed the Rap1 signaling pathway as a potential novel pathway charges. This article must therefore be hereby marked advertisement in accordance implicated in CRLF2r Ph-like ALL. The Rap1 signaling pathway is with 18 U.S.C. Section 1734 solely to indicate this fact. known for its role in regulating cell adhesion through its effect on integrin and cadherin proteins (33), and it has been implicated in Received November 12, 2019; revised June 30, 2020; accepted August 4, 2020; cancer invasion and metastasis (34). Specifically in hematologic published first August 14, 2020.

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Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-like ALL

Keith C.S. Sia, Ling Zhong, Chelsea Mayoh, et al.

Mol Cancer Res Published OnlineFirst August 14, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-1098

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