Targeting ALK in pediatric RMS does not induce antitumor activity in vivo

Abstract

Purpose: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor.

Methods: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo.

Results: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo.

Conclusions: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

Keywords: ALK; ALK inhibitors; Crizotinib; Patient-derived xenografts; Rhabdomyosarcoma.

Fig 1. Expression of ALK, MET and ROS1 in primary pediatric patient tumor samples, RMS cell lines and PDX as determined by RNAseq, qPCR and western analyses

A, B and C. The box plots on the left hand side represent results obtained from RNAseq datasets using 26 ERMS and 14 ARMS patient tumor specimens. The bar charts on the right hand side represent qPCR analyses of RNA isolated from RMS cell lines and PDX. Values represent relative delta ΔCt numbers using either TBP or ACTB as a control, with the LHCN-M2 result arbitrarily set to 1. D. Western analysis of ALK expression in RMS cell lines E. Western analysis of ALK expression in selected patient-derived intramuscularly grown RMS xenografts. This data has been presented as supplementary data previously [18], but is included here for comparative purposes. For all gels, size markers are in kDa. F. Growth inhibition curves for selected cell lines when treated with NVP-TAE684. A representative example is shown to demonstrate the high R² values (typically greater than 0.96) for the curve fits for each individual data set. For clarity, error bars are not shown.

Fig 2. Cell proliferation curves and transfection efficiencies for cell lines following transfection with siRNA targeting ALK

A. Cell proliferation curves for LHCN-M2, Rh3, Rh28 and SJRHB012_Y cells following transfection with siRNA directed towards ALK. Control samples were transfected with a scrambled siRNA. B. Phase contrast (upper panel) and fluorescence images (lower panel) of cells either 24hr (LHCN-M2, Rh3 Rh28) or 72hr (SJRHB012_Y) after transfection. A GFP-tagged control RNA was used to monitor transfection efficiency. C. Western analysis of ALK protein in cells following siRNA transfection. An image for LHCN-M2 is not shown since ALK expression is undetectable in these cells using this approach (see Figure 1D).

Fig 3. Xenograft growth curves and Kaplan-Meier curves for mice bearing SJRHB012_Y tumors when given ALK inhibitors either as single agents or when combined with VAC

A. Individual photon flux curves (as monitored by bioluminescent imaging) for mice bearing SJRHB012_Y xenografts grown in the thigh muscle of CD1 mice. Animals were orally dosed daily at the MTD for crizotinib (red line – CRI), NVP-TAE684 (blue line – TAE) or LDK-378 (green line – LDK) for 20 days. Data points represent the sum of the values for bilaterally implanted tumors following imaging on the dorsal side of the animal. B. Animal survival curves for mice harboring SJRHB012_Y xenografts following daily dosing with ALK inhibitors. The labeling of the curves is identical to that depicted in panel A. Median survival (in days) and statistical significance versus the control untreated group are indicated on the legend. C and D. Representative bioluminescence images of cohorts of mice bearing intramuscular SJRHB012_Y RMS xenografts. Panel C depicts the control group at day 27 and panel D is the crizotinib-treated animals at the same time point. E. Concentrations of crizotinib in plasma, muscle and tumor for mice bearing SJRHB012_Y RMS xenografts. Crizotinib (150mg/kg) was given orally for three consecutive days and samples harvested 4 hours following the final dose. Samples from the same animals are connected by the lines. F. Individual photon flux curves for mice bearing SJRHB012_Y RMS xenografts when treated with VAC alone (orange line – VAC), or when combined with oral daily dosing of crizotinib (red line – CRI), NVP-TAE684 (blue line – TAE) or LDK-378 (green line – LDK) for 20 days. Data points represent the sum of the values for bilaterally implanted tumors following imaging on the dorsal side of the animal. G. Animal survival curves for mice harboring SJRHB012_Y RMS xenografts following VAC therapy and daily dosing with ALK inhibitors. Curves are labeled in an identical manner to that described in panel B. Median survival (in days) and statistical significance vs the VAC alone group are indicated on the legend.