Targeting TSLP-Induced Tyrosine Kinase Signaling Pathways in CRLF2-Rearranged Ph-Like ALL Keith C.S
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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 con- trast 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 Ó2020 American Association for Cancer Research. targetable TKs and signaling pathways that have not been AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst August 14, 2020; DOI: 10.1158/1541-7786.MCR-19-1098 Sia et al. 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 com- bination 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 com- bination 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 À À 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).