Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models

Abstract

Purpose: ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway.

Experimental design: We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX).

Results: Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth.

Conclusions: In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma.

Figure 1.

Lorlatinib is the most potent ALK inhibitor tested across ALK-mutant or ALK-amplified neuroblastoma cell lines and PDTC ex vivo models. 72-hour GI₅₀ values for ALK inhibitors. A, (i) TAE-684, (ii) crizotinib, (iii) ceritinib, (iv) alectinib, and (v) lorlatinib in a panel of neuroblastoma cell lines (M & A: mutant and amplified ALK; Mut.: mutant ALK; Amp.: amplified ALK; WT: wild-type ALK). Cell lines were grouped on the basis of the type of ALK alteration. Statistically significant differences were determined by one-way ANOVA with Bonferroni post hoc tests and paired testing versus WT. *, P ≤ 0.05; **, 0.01; ***, 0.001; ****, 0.0001. B, Neuroblastoma cell lines treated with 20 nmol/L of indicated inhibitor for 3 hours, and lysates subjected to ALK immunoassay for total ALK and pY1586 ALK. Statistically significant differences determined by paired, two-tailed t test. *, P ≤ 0.05; **, 0.01; ***, 0.001. C, Study of cell viability by analysis of the DSS after treatment with (i) crizotinib, (ii) ceritinib, (iii) alectinib, or (iv) lorlatinib in ALK-mutant and -amplified PDTC models. R1: replicate 1; R2 replicate 2. Statistically significant differences determined by unpaired, two-tailed t test. **, P ≤ 0.01.

Figure 2.

Lorlatinib treatment of Th-ALK^F1174L/MYCN tumor-bearing animals gives a survival advantage over vehicle control. A,In vivo analysis of a panel of ALK inhibitors including crizotinib, ceritinib, alectinib, and lorlatinib was carried out using the Th-ALK^F1174L/MYCN model. Tumor-bearing Th-ALK^F1174L/MYCN mice, were treated with the indicated inhibitor or its corresponding vehicle (veh.) over a 3-day interventional dosing schedule, and tumor volume change was monitored by MRI on day 0 and day 3. Each bar represents tumor volume change in an individual animal. Crizotinib versus vehicle ****, P < 0.0001. Alectinib versus vehicle **, P = 0.0021. Lorlatinib versus vehicle ***, P = 0.0002. B, Tumors from A were harvested for immunoassay testing of ALK and pY1586 ALK status. Alectinib versus vehicle ***, P = 0.0005. Lorlatinib versus vehicle **, P = 0.0037. C, Th-ALK^F1174L/MYCN animals were treated with lorlatinib twice daily versus vehicle control to assess survival. ****, P < 0.0001 according to log-rank (Mantel–Cox) test. D, Tumor volume was monitored by MRI. E, Representative abdominal MRI of an animal from the lorlatinib survival study versus vehicle control. Tumor outlined by white line.

Figure 3.

CAV chemotherapy in combination with lorlatinib leads to significantly enhanced survival in preclinical Th-ALK^F1174L/MYCN GEMM neuroblastoma. A, Survival study of lorlatinib, with and without CAV chemotherapy in Th-ALK^F1174L/MYCN and Th-MYCN (dashed line) GEMMs. Vehicle versus CAV and lorlatinib ***, P = 0.0006; CAV versus CAV and lorlatinib ***, P = 0.0005; lorlatinib versus CAV and lorlatinib ***, P = 0.0005 in the Th-ALK^F1174L/MYCN model according to log-rank (Mantel–Cox) test. B, MRI growth monitoring of Th-ALK^F1174L/MYCN tumors from the survival study in A. No statistical significance between groups. C, Heatmap of the expression of 34 selected genes across all the treatment groups. Noradrenergic (ADRN) and MES-like (mesenchymal cell–like). D, Mean of log₂ TMM (trimmed mean of M values) score for genes in ADRN (i) or MES (ii) panel across treatment groups.

Figure 4.

High baseline in vivo expression of ALK determines sensitivity to lorlatinib in an ALK-amplified PDX neuroblastoma model. A, Hematoxylin and Eosin (H&E) and ALK IHC staining of vehicle-treated tumors from indicated PDX models, treated for 3 days. GR-NB4 (B), IC-pPDX-112 (C), and HSJD-NB-012 (D) PDX models were treated with vehicle control, one dose of chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine), continuous lorlatinib, or chemotherapy and continuous lorlatinib combination. (i) Tumor volumes were monitored during treatment. (ii) pY1586/total ALK measured by immunoassay in tumor lysates taken at the end of the experiment (mean of two technical replicates; Veh.: vehicle; Lorla.: lorlatinib; C&L: CAV and lorlatinib). One-way ANOVA: B.ii: P < 0.0001; C.ii: not significant; D.ii: P = 0.0010 (iii). Immunoblots of signaling pathways downstream of ALK from tumor lysates as per (ii).

Figure 5.

Idasanutlin synergizes with ALK inhibitors in TP53 wild-type and ALK-aberrant neuroblastoma cell lines. CI values at each constant 1:1 ratio combination and average of CI values at ED50, ED75, and ED90 of idasanutlin in combination with TAE-684 (A) and alectinib (B). Functional analysis of idasanutlin in combination with TAE-684 and alectinib using sub-G₁ and cell-cycle phase distribution (C and D), and caspase 3/7 activity (E and F). IDA, idasanutlin; TAE, TAE-684; I&T, idasanutlin and TAE-684; ALE, alectinib; I&A, idasanutlin and alectinib. One-way ANOVA: (C): SHSY5Y *, P = 0.0441; NB1691 **, P = 0.0070; (D): SHSY5Y *, P = 0.0273; CLB-Ge2 *, P = 0.0230; (E): SHSY5Y **, P = 0.0043; NB1691 *, P = 0.0322; (F): SHSY5Y *, P = 0.0150; CLB-Ge2 ***, P = 0.0008.

Figure 6.

Combination effects of lorlatinib and idasanutlin in ALK-altered neuroblastoma PDX models. GR-NB4 (A), IC-pPDX-112 (B), and HSJD-NB-012 (C) PDX models were treated with vehicle control, idasanutlin, lorlatinib, or idasanutlin and lorlatinib combination. (i) Tumor volumes were monitored during treatment [with (A.ii) inset of GR-NB4 PDX up to day 10]. (A.iii, B.ii, and C.ii) pY1586/total ALK measured by immunoassay in tumor lysates taken at the end of the experiment (mean of two technical replicates). One-way ANOVA: A.iii: P = <0.0001; B.ii: P = 0.0331; C.ii: P = 0.0020.