Translational Strategies for Repotrectinib in Neuroblastoma

Neuroblastoma is the most common pediatric extracranial solid tumor with more than 650 cases in North America per year. Despite aggressive medical and surgical therapy, patients with high-risk disease have 5-year overall survival rates of 40% to 50% (1, 2), with even poorer outcomes in patients with relapsed/refractory disease. Therefore, development of novel treatment strategies is essential to improve survival rates among these patients. Preclinical work has demonstrated that aberrant activation and signaling through ALK, PI3K/AKT, MAPK, PLCγ, TrkB, Src/FAK, and JAK/STAT pathways contribute to deregulated proliferation and therapy resistance in neuroblastoma (3–9). Thus far, therapeutic strategies that target a singular pathway have not resulted in significant clinical benefit. For example, the administration of ALK inhibitors in vitro and in vivo demonstrates potent inhibition of cell growth in ALK-mutant cell lines and tumor growth inhibition in xenograft models (3, 10–14), but overall response rates in early-phase clinical studies of single-agent ALK inhibitors in patients with neuroblastoma range from 9% to 20% (15–17). Therefore, the evaluation of rational combination therapies incorporating novel agents that inhibit multiple relevant signaling pathways represents a treatment paradigm that may lead to more effective and durable treatment responses in high-risk tumors.

Repotrectinib (TPX-0005) is a novel macrocyclic multikinase inhibitor that has the potential to exploit several therapeutic vulnerabilities of neuroblastoma and was specifically designed to overcome multiple common mechanisms of therapy resistance. In addition to bypassing solvent front and gatekeeper mutations of ALK, ROS1, and TRK (18, 19), repotrectinib effectively inhibits multiple critical signaling pathways mediating proliferation, survival, angiogenesis, and drug resistance, including JAK2/STAT and Src/FAK, with low nanomolar kinase inhibitory profiles (18–22). Taken together, these distinct features position repotrectinib as a promising targeting agent that seeks to address and overcome mechanisms of therapy resistance encountered by earlier generation multikinase inhibitors.

The activity of repotrectinib has recently been demonstrated in ALK-mutant neuroblastoma cell lines and in a cell line–derived xenograft model (11). On the basis of the promising clinical activity in patients with ROS1-rearranged solid tumors and tyrosine kinase inhibitor–resistant tumors (23), and preclinical work demonstrating antitumor activity in solid tumors without ALK, ROS1, or NTRK aberrations (24, 25), we hypothesize that repotrectinib may have activity extending beyond ALK-mutant neuroblastoma. We further hypothesize that combining repotrectinib with cytotoxic chemotherapy may augment its activity. To evaluate the applicability of repotrectinib, singly and in combination with chemotherapy, we assembled and treated a clinically representative, diverse cohort of neuroblastoma patient derived xenograft (PDX) models representing the molecular spectrum of tumor phenotypes and provide preclinical rationale for further exploration within the context of a clinical trial.

Repotrectinib was provided by Turning Point Therapeutics. Irinotecan and temozolomide were obtained commercially from SelleckChem. Ensartinib (E543721) was obtained commercially from LKT Laboratories.

The neuroblastoma cells lines, SH-SY5Y (ATCC catalog no. CRL-2266, RRID:CVCL_0019), SK-N-DZ (ATCC catalog no. CRL-2149, RRID:CVCL_1701) and IMR-32 (ATCC catalog no. CCL-127, RRID:CVCL_0346) were obtained from the ATCC. Kelly (DSMZ catalog no. ACC-355 RRID:CVCL 2092) and LA-N-5 (DSMZ catalog no. ACC-673, RRID:CVCL_0389) were obtained from the German Collection of Microorganisms and Cell Cultures GmbH. NB-1 and GI-ME-N cell lines were provided by Dr. Frank Speleman. Cells were cultured in treated flasks in DMEM/F12 media (Corning) supplemented with 10% FBS (Corning), L-glutamine (Gibco), nonessential amino acids (Gibco), and antibiotic-antimycotic (Gibco) at 37°C and 5% CO₂. Cells were passaged twice weekly. Cell lines were obtained between 2014 and 2021 and cell line identities were verified by short tandem repeat (STR) profiling. Mycoplasma testing was performed prior to all in vitro drug screens using the MycoAlert-Plus kit (Lonza).

The assay was conducted in a 384-well format (Corning), and cells were seeded at optimized seed densities empirically determined for each cell line to achieve exponential growth during the assay period. Dose-response studies were performed using serial dilutions ranging from 100 μmol/L to 0.001 μmol/L with each concentration performed in triplicate. The high controls (HC) contained 1% DMSO (v/v) and the low controls (LC) contained 1 μmol/L of killer mix (ref. 26; combination of 6 cytotoxic drugs) in 1% DMSO (v/v). Cells were incubated at 37°C and 5% CO₂ and viability assayed with Alamar Blue after 96 hours of drug treatment. Fluorescence intensity was read on the Cytation 5 Cell Imaging Multi-Mode Reader (Biotek, RRID:SCR_019732). For the screening analysis, the Z factor was used to determine the quality of the assay, and is defined as 1–3 × (SD of the HC + SD of the LC/ HC average–LC average). Values between 0.5 and 1.0 represent an excellent performance of the assay, and a marginal assay shows values between 0 to 0.5 (27). Dose–response curves were fitted with a logistic 4-parameter sigmoidal equation using SigmaPlot 13.0 (Systat Software, Inc., RRID:SCR_003210). Percent inhibition was defined as (HC average–obtained value/HC average – LC average) × 100.

For the drug combination screen, the fixed ratio method (28) was used, where the concentrations of the drugs (drug 1: repotrectinib or ensartinib, drug 2: irinotecan, drug 3: temozolomide) were set at 1:1:0.1–1 the 50% growth inhibitory concentration (IC₅₀). Ratios were determined based on drug solubility characteristics at the specified concentrations. The IC₅₀ determined in the dose-response assays was used to establish the concentrations of each drug for the combination to assure that the IC₅₀ was approximately at the middle of the serial dilutions. 1.25-fold dilutions were done for a total of 23 points for the combinations in triplicate. The effect of the combination was assessed via the combination index (CI) obtained with the Chou and Talalay Method (29) using CalcuSyn Software version 2 (BIOSOFT, RRID:SCR_020251). The CI values are defined as synergistic if CI<1, additive if CI = 1, or antagonistic if CI>1. A semiquantitative CI annotation system (synergism, moderate synergism, slight synergism, nearly additive, slight antagonism, and moderate antagonism) has been defined previously (30).

For in vitro analysis, approximately 5×10⁶ cells were seeded in 10-cm culture dishes and FBS-starved for 24 hours prior to treatment. Cells were treated with either DMSO or repotrectinib (at concentrations of 10 nmol/L, 100 nmol/L, and 1 mmol/L) for 1 hour and then harvested with RIPA buffer (catalog no. 89901; ThermoFisher Scientific) supplemented with Halt Protease & Phosphatase Inhibitor Cocktail (catalog no. 1861284; ThermoFisher Scientific). For in vivo pharmacodynamic analyses, tumors were harvested 3 hours post-dose on the third day of treatment and tumors flash frozen in liquid nitrogen. Tumor lysates were obtained using RIPA buffer + protease/phosphatase inhibitor cocktail. Protein concentration was measured using the bicinchoninic acid (BCA) assay (catalog no. 23210; ThermoFisher Scientific) and 20 μg of total protein was analyzed on SDS-PAGE gels and transferred to nitrocellulose membrane using standard methods. Membranes were probed with the following primary antibodies: phospho-ALK (Y1278) (catalog no. 6941; Cell Signaling Technologies, CST, RRID:AB_10860598), phospho-ALK (Y1586; catalog no. 3348; CST, RRID:AB_659803), Pan Trk (catalog no. 92991; CST, RRID:AB_2800196), phospho-TrkA/TrkB (catalog no. 4621; CST, RRID:916186), FAK (catalog no. 13009; CST, RRID:AB_2798086), phospho-FAK (catalog no. 3283; CST, RRID:AB_2173659), Src (catalog no. 2109; CST RRID:AB_2106059), phospho-Src (catalog no. 2101; CST, RRID:AB_331697), PI3K (catalog no. 4257; CST, RRID: AB_659889), AKT (catalog no. 75692; CST, RRID:AB_2716309), phospho-AKT (catalog no. 4060; CST, RRID:AB_2315049), STAT3 (catalog no. 9139; CST, RRID:AB_331757), phospho-STAT3 (catalog no. 9131; CST, RRID:AB_331586), MEK1/2 (catalog no. 4694; CST, RRID:AB_10695868), phospho-MEK1/2 (catalog no. 9154; CST, RRID:AB_2138017), Erk1/2 (catalog no. 9102; CST, RRID:AB_330744), phospho-Erk1/2 (9101; CST, RRID:AB_331646), RPS6 (catalog no. 2217; CST, RRID:AB_331355), phospho-RPS6 (catalog no. 4858; CST, RRID:AB_916156), phospho-ALK (Y1604; catalog no. ab62185; Abcam, RRID:AB_955656), phospho-PI3K (catalog no. ab138364; Abcam, RRID:AB_2892208), ALK (catalog no. 398791; Santa Cruz Biotechnology, RRID:AB_2889357), tubulin (catalog no. T9026; Sigma, RRID:AB_477593). Secondary antibodies were obtained from LI-COR: anti-mouse IgG (catalog no. 926–68072, RRID:AB_10953628) and anti-rabbit (catalog no. 926–32213, RRID:AB_621848). Signal detection and image capturing were performed using the LI-COR Odyssey scanner system.

All mice were maintained under barrier conditions and experiments were conducted in accordance with and with the approval of Memorial Sloan Kettering Cancer Center (MSKCC) Institutional Animal Care and Use Committee (IACUC; protocol #16–08–011). Patient-derived tumor tissue to generate PDX models was obtained under the MSKCC Institutional Review Board (IRB)–approved protocols #17–387 and #06–107 with written informed consent provided by the subject or legally authorized representative. PDX mouse models were established by implanting tumor cells subcutaneously into nonobese diabetic/severe combined immunodeficiency IL2Rγ null, hypoxanthine phosphoribosyltransferase (HPRT)–null (NSGH) mice (Jackson Labs, IMSR catalog no. JAX:012480, RRID: IMSR_JAX:012480). Two mice of each model were enrolled into each treatment arm as a preliminary screening single mouse trial (2 mice per model per treatment) adapted from Gao and colleagues (31) Once the tumor reached a volume of 150 to 200 mm³, mice were assigned to treatment groups using block randomization with 2 animals per group. Mice were treated in 1 of 3 groups for 4 weeks (treatment 5 days on, 2 days off for all arms): (i) repotrectinib 20 mg/kg via oral gavage twice daily, (ii) ensartinib 25 mg/kg orally twice daily, and, (iii) vehicle [0.5% carboxymethylcellulose (CMC)/1% Tween-80] orally twice daily. Tumors were measured by caliper measurement twice weekly and tumor volume (TV) was calculated as follows: TV = width² × ½ length. Aligned with clinical response criteria (32, 33), responses were categorized as complete response (CR, >95% reduction from baseline or no measurable tumor), partial response (PR, >50% reduction), stable disease (SD, <50% reduction but not more than 100% increase), or progressive disease (PD, >100% increase). Overall response rate was defined as the total of responses categorized as CR or PR. To account for variable tumor growth rates across different PDX models, the timepoint at which each individual model's tumor volume change was analyzed corresponded to the day of treatment failure (>100% increase in tumor volume relative to baseline) in each respective model's vehicle-treated animals.

For xenograft studies using combination therapy, a minimum of 3 mice were included in each of four treatment arms: (i) repotrectinib 15 mg/kg orally twice daily (5 days on, 2 days off) continuously throughout treatment cycle; (ii) irinotecan 6 mg/kg/dose via intraperitoneal injection and temozolomide 25 mg/kg/dose orally daily on days 1 to 5 and 15 to 29 of each 28-day cycle; (iii) repotrectinib 15 mg/kg orally twice daily (5 days on, 2 days off) throughout 28-day treatment cycle + irinotecan 6 mg/kg/dose intraperitoneally and temozolomide 25 mg/kg/dose orally daily on days 1 to 5 and 15 to 29 of each 28-day cycle; and (iv) vehicle (0.5% CMC/1% Tween-80) orally twice daily. Mean relative tumor volumes (RTV) were calculated for vehicle control (C) and each treatment arm (T). The drug associated with mean RTV T/C ratio of <15% are considered highly active, <45% but >15% are considered to have intermediate activity, and >45% are considered to have low levels of activity (34).

For in vivo statistical analysis, the Mann–Whitney–Wilcoxon method was used to evaluate differences in distribution of tumor volume between treatment groups. Vardi test was used to evaluate difference in AUC between treatment groups. Event-free survival (EFS) was defined as the percentage of mice that survived at any given timepoint without an event. Accounting for inter-operator variability of caliper measurements (35) and consistent with clinical treatment response definitions (33), an event was defined as a 100% increase in tumor volume relative to baseline measurements or death. Kaplan–Meier survival curves were compared using the log-rank test. Statistical analysis was performed using R software (v3.5.0). Waterfall plots and tumor volume curves for in vivo analysis were generated with GraphPad Prism [v8.4.1 (RRID:SCR_002798)]. Statistical significance was defined as P values < 0.05.

To determine the antitumor activity of repotrectinib in vivo, we evaluated its antitumor effect in several pediatric PDX models (Table 1), utilizing a modification of the Single Mouse Trial (SMT) design (31). Eight discrete neuroblastoma models were included in the therapeutic study. For each model, 2 animals were evaluated on each treatment arm (n = 16 per treatment group). When evaluating the change in tumor volume from baseline between treatment groups, there was a statistically significant difference between repotrectinib and vehicle treatment (median tumor volume change of 14% and 149%, respectively, P = 0.005) and ensartinib versus vehicle (73% and 149%, respectively, P = 0.005; Fig. 1A). In comparing the repotrectinib and ensartinib treatment cohorts, there was a trend toward reduced tumor volume in the repotrectinib group, although statistical significance was not reached at the P = 0.05 threshold (14% and 73%, P = 0.12; Fig. 1A). When stratifying change in relative tumor volume by treatment arm based on ALK status, ALK-mutant models treated with ensartinib or repotrectinib had statistically significantly reduced change in relative tumor volume compared with the vehicle arm (P = 0.005 and P = 0.014, respectively). However, ALK wild-type (WT) models treated with repotrectinib showed greater relative tumor volume change from baseline compared with vehicle (P = 0.041), in contrast to those treated with ensartinib compared with vehicle (P = 0.10; Supplementary Fig. S1). Furthermore, EFS was analyzed, and repotrectinib yielded a significant survival advantage when compared with the vehicle or ensartinib groups in the total neuroblastoma cohort (n = 8 models) and the ALK-mutant neuroblastoma cohort (n = 5 models; P < 0.05, via log-rank test in all treatment comparisons; Fig. 1B and C). In the ALK WT cohort, the repotrectinib treatment arm also had improved EFS compared with vehicle (P = 0.04, via log–rank) supporting activity of repotrectinib in both ALK-mutant and WT models.

We evaluated the antiproliferative effect of repotrectinib and ensartinib in 7 neuroblastoma cell lines (Supplementary Table S1). Treatment of cells with repotrectinib or ensartinib demonstrated a dose-dependent antiproliferative effect for both drugs. Repotrectinib and ensartinib effects were most pronounced in NB-1 (ALK amplified) and LAN-5 (ALK R1275Q) and least potent in SK-N-DZ (ALK WT, MYCN amplified, TP53 mutant; Table 2; Supplementary Fig. S2). When stratifying the IC₅₀ of repotrectinib by various genomic factors including ALK status (presence of mutation or amplification), MYCN status (presence of amplification) or TP53 status (presence of mutation), there was no clear segregation of sensitivity based on molecular alterations (Fig. 2A). The IC₅₀ of irinotecan and temozolomide used as single agents was also calculated in all cell lines in anticipation of combination therapy (Table 2; Supplementary Fig. S2). Temozolomide IC₅₀ values were high (114–1,500 μmol/L) and reflect the requirement for autocatalysis of temozolomide in culture medium to its active metabolite, MTIC (36). However, the observed IC₅₀ values of temozolomide are similar to previously determined values in the literature (37).

To assess for combinatorial activity of repotrectinib with chemotherapy, the fractional effect points (fraction of cells affected at each respective treatment dose) were plotted against the CI for each unique cell line and treatment condition. The combination index defines the combination of two or more drugs as synergistic (CI < 1), additive (CI = 1), or antagonistic (CI > 1). Targeted inhibition (with repotrectinib or ensartinib) in combination with irinotecan and temozolomide demonstrated a robust cytotoxic effect (additive to synergistic) in all ALK-aberrant cell lines and two ALK WT cell lines. This is effectively demonstrated by evaluating the CI values generated in the interquartile range (25%–75%) of the fractional effect points for each cell line and treatment condition (Fig. 2B and C; Supplementary Table S2). For example, when evaluating Kelly, the CI of irinotecan and temozolomide treatment ranges from 0.868–1.194 (slight synergism to slight antagonism). With the addition of repotrectinib and ensartinib, CI values range from 0.699–0.930 and 0.570–0.73, respectively, suggesting a nonantagonistic effect range (all CI <1). The nonantagonistic effect of targeted inhibition plus chemotherapy holds true in nearly every cell line, treatment, and fractional effect combination evaluated in ALK-aberrant cell lines as well as 2 ALK WT cell lines (SK-N-DZ and GI-ME-N). Overall, these findings suggest a cooperative effect when combining growth inhibitory agents with chemotherapy in vitro across a phenotypically diverse panel of neuroblastoma cell lines.

In order to evaluate target engagement and downstream signaling inhibition by repotrectinib in the most sensitive (NB-1) and least sensitive (SK-N-DZ) neuroblastoma cell lines, we treated cells with increasing concentrations of repotrectinib and evaluated changes in signaling pathways that have been shown to be targeted by repotrectinib (ALK/p-ALK, Trk/p-Trk, FAK/p-FAK, Src/p-Src, PI3K/p-PI3K, AKT/p-AKT, STAT3/p-STAT3, MEK 1/2/p-MEK1/2, Erk1/2/p-Erk1/2, S6/p-S6; Fig. 3A–C). Both cell lines showed reductions in p-FAK and p-Src at the highest repotrectinib treatment concentration (1 μmol/L). While SK-N-DZ showed a more marked reduction in p-Trk, NB-1 showed clear dose-dependent abrogation of p-PI3K, p-AKT, p-STAT3, pMEK1/2, pErk1/2, and p-S6 expression compared with SK-N-DZ. These findings suggest that more effective downregulation of multiple pathways implicated in cell growth and proliferation may contribute to increased sensitivity to repotrectinib. As the single agent IC₅₀ of repotrectinib in SK-N-DZ (2.3 μmol/L) is higher than that of NB-1, treatment at the maximal treatment concentration (1 μmol/L) may have precluded demonstration of pathway inhibition.

To further assess pharmacodynamic efficacy of repotrectinib, we utilized an ALK-amplified (ALK R1275Q, ALK amplification, MYCN amplification) neuroblastoma PDX model (MSKNBL-40352). Tumors were collected after 6 doses (3 days) of repotrectinib treatment, and immunoblot analysis of tumors revealed a similar pattern of ALK, PI3K/AKT, and MAPK pathway inhibition (Supplementary Fig. S3, Fig. 3D).

To evaluate the comparative antitumor activity of chemotherapy alone versus in combination with repotrectinib in vivo, we evaluated an ALK-mutant neuroblastoma PDX model (MSKNBL-30595, MYCN amplified, ALK F1174V, TERT promoter mutation) and an ALK WT neuroblastoma PDX model (MSKNBL-82180, MYCN WT, ALK WT, ATRX E553*). The two neuroblastoma PDX models were selected as they showed treatment resistance to repotrectinib in the monotherapy therapeutic studies (Supplementary Fig. S3). We enrolled mice onto one of four treatment arms: (i) repotrectinib, (ii) irinotecan and temozolomide, (iii) repotrectinib plus irinotecan and temozolomide, and (iv) vehicle control. All treatments were tolerated well with no signs of systemic toxicity or weight loss (Supplementary Fig. S4). In the ALK-mutant model (n = 10 per treatment arm), when comparing best response (% tumor reduction from baseline) after 2 cycles of chemotherapy, the repotrectinib plus chemotherapy cohort had significantly reduced tumor volume compared with all other treatment groups, including chemotherapy alone (P < 0.001, Mann–Whitney–Wilcoxon test; Fig. 4A). Furthermore, when comparing average tumor volume throughout the treatment interval, repotrectinib plus chemotherapy was superior to treatment with chemotherapy alone (P = 0.025, Vardi test; Fig. 4B). When evaluating the role of repotrectinib plus chemotherapy in the ALK WT model (n = 3 per treatment arm with exception of irinotecan/temozolomide arm, n = 4, due to rolling enrollment of study animals), both irinotecan/temozolomide and repotrectinib plus irinotecan/temozolomide induced rapid CRs (Fig. 4C). Median time to complete response in the repotrectinib plus chemotherapy arm was 28 days (range 21–38 days) compared with 44 days in the chemotherapy-alone arm. RTVs were calculated for vehicle C and each T at day 18 (last day with measurable tumor in all mice). The mean RTVs were 74%, 4.8%, and 4.3% in the repotrectinib, chemotherapy, and repotrectinib plus chemotherapy arms respectively.

Due to its macrocyclic structure, small size, and broad kinase inhibitory profile, repotrectinib may provide additional benefit compared with earlier generation kinase inhibitors trialed in neuroblastoma and may augment treatment response when used in combination with chemotherapy. In preclinical studies published to date, repotrectinib has shown potent activity in models characterized by ALK, ROS, and NTRK fusions (19), with in vitro cytotoxicity observed in several neuroblastoma cell lines, and in vivo antitumor effect in one cell-line derived neuroblastoma xenograft model (11). In our study, we extend these findings by confirming the antitumor effects of repotrectinib across a genomically heterogeneous cohort of PDX models that recapitulate the diverse genomic landscape of neuroblastoma seen in the clinic. We also explore the applicability of repotrectinib in ALK-mutant and ALK WT disease and demonstrate potential therapeutic benefit of repotrectinib in combination with a clinically relevant chemotherapy backbone for neuroblastoma.

Our in vivo studies evaluating repotrectinib monotherapy included PDX models with variable mutational status of the following clinically relevant genes in neuroblastoma: MYCN, ALK, TERT, and ATRX. This diverse genotypic representation of neuroblastoma models underscores the applicability and clinical translatability of our preclinical results based upon the prevalence of recurrent molecular alterations observed in patients with neuroblastoma. With regard to tumor-growth inhibition in pediatric neuroblastoma PDXs, repotrectinib monotherapy slowed tumor growth significantly compared with the vehicle control (P = 0.005), with the tested cohort inclusive of ALK-WT and ALK-mutant models. Repotrectinib treated PDXs also had improved EFS compared with the other two treatment groups (P < 0.05). However, despite promising preclinical findings with repotrectinib monotherapy, the overall observed clinical response rates are suboptimal and suggests the need for combination therapy with additional drugs, such as cytotoxic agents, for improved antitumor control.

When evaluating repotrectinib monotherapy in vitro, comparable cytotoxic activity is seen with its comparator in class, ensartinib, and single-agent IC_50′s for repotrectinib did not segregate based on ALK, MYCN, or TP53 mutational status of the cell lines tested. While the combination of growth inhibitory agents, such as repotrectinib, and cell-cycle specific chemotherapy has the potential to be antagonistic, our findings do not demonstrate a significant antagonistic effect, and provide rationale for further exploring this multiagent regimen. In combining repotrectinib with chemotherapy in vitro, additive to synergistic cytotoxicity was noted across almost all neuroblastoma cell lines tested. Interestingly, the repotrectinib single-agent IC₅₀ of each cell line did not directly correlate with the degree of additivity to synergy seen in combination with chemotherapy (irrespective of ALK mutational status). This suggests that there are likely complex signaling interactions contributing to sensitivity or resistance to combination therapy dependent on each cell line's specific genotype that need to be further elucidated. Overall, our results are comparable with those of Krytska and colleagues who demonstrated that crizotinib synergizes with cyclophosphamide and topotecan in vitro and in vivo (38). Similar to Krytska and colleagues, we evaluated a chemotherapy backbone with known antineuroblastoma activity. However, our findings show that the additive to synergistic effect of repotrectinib plus chemotherapy was not limited to ALK-aberrant, WT TP53 models. In addition to the ALK-aberrant models with functional p53, we show evidence of additivity to synergism in two TP53-mutant cell lines including an ALK WT cell line (SK-N-DZ). These findings suggest that non–ALK-dependent signaling (JAK2/STAT, Src/FAK) may be contributing to the antiproliferative effect. Taken together, these results, along with the applicability of irinotecan and temozolomide as a rational salvage regimen in patients with neuroblastoma and several other solid tumors, support the clinical translation of this novel drug combination (39, 40).

The cooperative cytotoxicity noted in vitro with repotrectinib plus chemotherapy treatment was recapitulated in vivo. The MSKNBL-30595 PDX model (MYCN amplified, ALK F1174V, TERT promoter mutation) which demonstrated rapid progression when treated with repotrectinib monotherapy, was noted to have statistically significant reduced tumor volume in the repotrectinib plus irinotecan/temozolomide treatment arm compared with chemotherapy alone when evaluating the best response after two cycles of therapy (Fig. 4A). Additionally, the repotrectinib plus chemotherapy arm had significantly lower average tumor volume throughout the therapeutic study (Fig. 4B). While the sample size was small when evaluating the effect of repotrectinib in combination with chemotherapy in the MSKNBL-82180 model (MYCN WT, ALK WT, ATRX mutant), our results show rapid and durable CRs with ongoing disease control beyond completion of scheduled drug treatments, as well as a trend toward faster time to response with the addition of multikinase inhibition to chemotherapy.

Limited published clinical data are available regarding the role of multikinase inhibitor therapy (monotherapy; refs. 41, 42; or in combination with chemotherapy) in neuroblastoma. There has been a more robust experience with ALK inhibitor monotherapy in this population, but so far this has not yet been applied as a tractable therapeutic option likely due to the upregulation of compensatory signaling pathways seen in preclinical models. These mechanisms include AXL phosphorylation potentiating MAPK signaling and the development of an epithelial-to-mesenchymal transition (EMT) phenotype leading to treatment resistance, among other mechanisms (43). Repotrectinib inhibits the JAK2/STAT and Src/FAK pathways (of which both can interact with downstream MAPK signaling; refs. 18, 19), and suppressed activation of Src, FAK, MEK1/2, and Erk1/2 were demonstrated in a repotrectinib treated ALK-aberrant cell line and xenograft model. Therefore, multikinase inhibition achieved with repotrectinib may contribute to broader efficacy across neuroblastoma models resistant to earlier generation inhibitors (e.g., ALK 1174 L mutations with conferred crizotinib resistance). Other preclinical studies confirm that inhibition of the JAK/STAT and Src/FAK pathways can abrogate tumor growth either as single-agent therapy or in combination with other targeted inhibitors through reestablishing sensitivity in the setting of on-treatment resistance (44). For example, in preclinical models of KRAS-mutant non–small cell lung cancer (i.e., without ALK, ROS1, or NTRK aberrations), repotrectinib treated models demonstrated tumor growth inhibition with notable downregulation of p-FAK, p-Src, and p-Erk by immunoblotting. Further, synergistic activity was seen when repotrectinib was administered in combination with trametinib, likely due to targeting of the upregulated compensatory signaling pathways that promote drug resistance (24). When considering the role of the Src pathway in neuroblastoma specifically, the Src/ABL inhibitor, bosutinib, had potent antineuroblastoma activity in vitro and in vivo, and enhanced the effect of conventional cytotoxic chemotherapy (45). Based on these preclinical findings, it is reasonable to hypothesize that broad, multikinase pathway inhibition by repotrectinib contributes to therapeutic efficacy in neuroblastoma xenograft models and warrants future investigation.

In summary, repotrectinib is a promising next generation multikinase inhibitor with activity directed toward a number of signaling pathways implicated in neuroblastoma biology. This novel agent has activity in a wide array of genotypically distinct neuroblastoma models irrespective of ALK, MYCN, or TP53 status. When repotrectinib is combined with chemotherapy in vitro and in vivo, superior antiproliferative and antitumor effect is demonstrated most notably in ALK-aberrant tumors. There is an ongoing multicenter, international, first in pediatrics phase I/II trial evaluating repotrectinib monotherapy in pediatric patients with relapsed and refractory solid tumors (NCT04094610). Further work is needed to better define the mechanisms of treatment responses and resistance in order to identify populations that may enrich for response to repotrectinib, such as ALK-mutant neuroblastoma. Based on the promising preclinical data presented, a subsequent clinical trial has been developed to evaluate the clinical utility of repotrectinib in combination with cytotoxic chemotherapy in pediatric patients.

T.J. O'Donohue reports nonfinancial support from Turning Point Therapeutics during the conduct of the study; and nonfinancial support and other support from Turning Point Therapeutics outside the submitted work. A.L. Kung reports personal fees from Emendo Biotherapeutics, Karyopharm Therapeutics, Imago BioSciences, DarwinHealth; and personal fees from Isabl Technologies outside the submitted work; in addition, A.L. Kung has a patent for whole genome analysis licensed to Isabl Technologies; and a patent for engineered cell lines licensed to Covance. No disclosures were reported by the other authors.

T.J. O'Donohue: Conceptualization, data curation, formal analysis, supervision, investigation, visualization, methodology, writing–original draft, writing–review and editing. G. Ibáñez: Formal analysis, investigation, visualization, methodology, writing–review and editing. D. Ferreira Coutinho: Data curation, formal analysis, investigation, visualization, methodology, writing–review and editing. A. Mauguen: Formal analysis, visualization, writing–review and editing. A. Siddiquee: Investigation, methodology. N. Rosales: Investigation, methodology. P. Calder: Investigation. A. Ndengu: Investigation, writing–review and editing. D. You: Methodology, project administration. M. Long: Investigation. S.S. Roberts: Conceptualization, supervision, writing–review and editing. A.L. Kung: Conceptualization, resources, supervision, funding acquisition, methodology, writing–review and editing. F.S. Dela Cruz: Conceptualization, supervision, methodology, writing–review and editing.

This research was supported by the Gold Ribbon Riders and Cancer Center Support grant P30 CA008748. T.J. O'Donohue is partially supported by the NIH/NCATS grant number UL1-TR-002384, Swim Across America Young Investigator Award, Kristen Ann Carr Fund, Margaux's Miracle, and TeamConnor. F.S. Dela Cruz is partially supported by The Paulie Strong Foundation, The Grayson Fund, and The Willens Family Fund. We would like to thank Turning Point Therapeutics for supplying repotrectinib for these preclinical studies, Dr. Frank Speleman for providing the NB-1 and GI-ME-N cell lines, and Joseph Olechnowicz (Memorial Sloan Kettering Department of Pediatrics, Senior Editor) for his editorial assistance.

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