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Cabozantinib for neurofibromatosis type 1–related plexiform neurofibromas: a phase 2 trial

Abstract

Neurofibromatosis type 1 (NF1) plexiform neurofibromas (PNs) are progressive, multicellular neoplasms that cause morbidity and may transform to sarcoma. Treatment of Nf1fl/fl;Postn-Cre mice with cabozantinib, an inhibitor of multiple tyrosine kinases, caused a reduction in PN size and number and differential modulation of kinases in cell lineages that drive PN growth. Based on these findings, the Neurofibromatosis Clinical Trials Consortium conducted a phase II, open-label, nonrandomized Simon two-stage study to assess the safety, efficacy and biologic activity of cabozantinib in patients ≥16 years of age with NF1 and progressive or symptomatic, inoperable PN (NCT02101736). The trial met its primary outcome, defined as ≥25% of patients achieving a partial response (PR, defined as ≥20% reduction in target lesion volume as assessed by magnetic resonance imaging (MRI)) after 12 cycles of therapy. Secondary outcomes included adverse events (AEs), patient-reported outcomes (PROs) assessing pain and quality of life (QOL), pharmacokinetics (PK) and the levels of circulating endothelial cells and cytokines. Eight of 19 evaluable (42%) trial participants achieved a PR. The median change in tumor volume was 15.2% (range, +2.2% to −36.9%), and no patients had disease progression while on treatment. Nine patients required dose reduction or discontinuation of therapy due to AEs; common AEs included gastrointestinal toxicity, hypothyroidism, fatigue and palmar plantar erythrodysesthesia. A total of 11 grade 3 AEs occurred in eight patients. Patients with PR had a significant reduction in tumor pain intensity and pain interference in daily life but no change in global QOL scores. These data indicate that cabozantinib is active in NF1-associated PN, resulting in tumor volume reduction and pain improvement.

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Fig. 1: Cabozantinib reduces PN tumor burden in Nf1-mutant mice.
Fig. 2: MIB–MS profiling reveals cabozantinib target kinases in PN tumors of Nf1-mutant mice.
Fig. 3: Clinical trial schema and CONSORT diagram.
Fig. 4: Participant responses to cabozantinib.

Data availability

Raw MS files pertaining to Fig. 2 and Extended Data Fig. 4 and 5 are available in the PRIDE database (https://www.ebi.ac.uk/pride/archive/projects/PXD019138). log2-transformed LFQ MIB binding values are provided. Raw data for circulating progenitor cell and cytokine analysis associated with Extended Data Fig. 10 and Supplementary Tables 10 and 11 are also provided in Supplementary Data 1. Source data are provided with this paper.

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Acknowledgements

This work was supported by the USAMRMC, through the Neurofibromatosis Research Program (NFRP), Clinical Consortium Award (CCA), funding opportunity no. W81XWH-11-NFRP-CCA, under award no. W81XWH-12-01-0155. Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the US Army. This research was further supported by a Developmental and Hyperactive Ras Tumor SPORE funded through the NIH/NCI (U54-CA196519-04) and Exelixis. S.D.R. is a fellow in the Pediatric Scientist Development Program supported by award no. K12-HD000850 from the Eunice Kennedy Shriver National Institute of Child Health and Human Development and the Francis S. Collins Scholars Program in Neurofibromatosis Clinical and Translational Research funded by the Neurofibromatosis Therapeutic Acceleration Program (2004757180). We thank the Multiplex Analysis Core at the Indiana University Melvin and Bren Simon Cancer Center for providing support in analyzing samples and interpretation of data. We thank A. Horvai (Department of Pathology, University of California San Francisco) for external histopathological review of murine PN specimens, A. Masters (Laboratory Director of the Clinical Pharmacology Analytical Core at the Indiana University Melvin and Bren Simon Cancer Center) for PK profiling of cabozantinib in mouse peripheral blood and nerve tissue specimens and E. Sims (Angio BioCore at the Indiana University Melvin and Bren Simon Cancer Center) for multiparametric flow cytometry analysis of peripheral blood samples from study participants. We thank K. Cole-Plourde, E. Davis and C.S. Powell from the NF Clinical Trials Operations Center (University of Alabama Birmingham, AL) for supporting the clinical trial. We thank K. Shannon for his helpful comments, discussions and reading of the manuscript.

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Contributions

D.W.C. and S.D.R. conceptualized and designed the studies evaluating cabozantinib in the preclinical NF1 mouse model. W.K.B. conducted the in vivo preclinical therapeutic studies. L.J. microdissected tumor-bearing nerve tissues from experimental mice and measured proximal nerve volumes. A.E.S. performed immunohistochemical stains and conducted histopathological evaluation of all tumor specimens with assistance from S.D.R. S.P.A. and G.L.J. generated and analyzed the MIB–MS data from preclinical specimens. S.D.R., Y.H., S.-J.P. and J.A.M. performed lineage-specific validation of cabozantinib targets in cell culture assays and by western blot. S.D.R. and A.E.A. evaluated sAXL in both preclinical and participant samples. S.D.R. reviewed the preclinical data and generated the corresponding text and figures. M.J.F., C.-S.S. and J.O.B. designed and chaired the clinical trial. K.A.R., B.C.W. and B.R.K. were part of the clinical trial study team. M.J.F., C.-S.S., R.J.P., J.C.A., N.J.U., S.G., D.H.G., S.R.P., T.R. and B.R.K. enrolled participants and supervised the clinical research teams at the institutions that enrolled participants. B.R.K. directs the NF Clinical Trials operations center. P.L.W. led the PRO evaluation. E.D. performed the central review of all imaging studies. J.A.M. assisted with measurement of proangiogenic CHSPCs and performed in vitro ECFC assays. K.-B.V. processed and banked plasma samples for cytokine analysis and designed experiments for cytokine multiplex assays performed by the core facility. C.T.R. and G.R.C. completed the statistical analysis and modeling of the clinical data. C.T.R. managed all clinical data and generated the clinical figures. C.-S.S., M.J.F., A.E.A., J.O.B. and P.L.W. analyzed and interpreted the clinical trial data. M.J.F., S.D.R., A.E.A., J.O.B., D.W.C., C.-S.S. and P.L.W. drafted the manuscript. All authors reviewed, commented on and approved of the manuscript.

Corresponding authors

Correspondence to Jaishri O. Blakeley or D. Wade Clapp.

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Competing interests

The authors declare the following competing interests: C.-S.S. is currently employed at Merck Research Laboratories (MRL) within Merck and Co. in Late Stage Oncology Clinical Development and is a consultant for the Selumetinib NF program at MRL. P.L.W. has holdings in Bristol-Myers Squibb under the amount allowable by the NIH. The remaining authors have no competing interests to declare.

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Extended data

Extended Data Fig. 1 Pharmacokinetics of cabozantinib in Nf1flox/flox;PostnCre mice.

a, Concentration-time profiles of cabozantinib in plasma and nerve tissue samples from n = 3 mice at each time. Plasma samples were measured at 1, 2, 4, 8, and 24 hours, while tissue samples were measured at 4 and 24 hours after a single 15 mg/kg oral gavage dose of cabozantinib. The error bars indicate standard deviations. b, Pharmacokinetic outcomes of cabozantinib in n = 3 mice administered a single 15 mg/kg oral gavage dose. All values are presented as the arithmetic mean. c, Chromatography of cabozantinib (top) at the lowest limit of quantification, 3 ng/mL, and the internal standard, temazepam (bottom). d, Chromatography of cabozantinib (top) and the internal standard, temazepam (bottom). The filled peaks are the analyte of interest.

Source data

Extended Data Fig. 2 Representative tumors from vehicle and cabozantinib treated Nf1flox/flox;PostnCre mice.

H&E stained photomicrographs of plexiform neurofibroma tumors in the vehicle a, and cabozantinib b, treated cohorts. Magnification are as denoted by the scale bars. The experiment was repeated three times independently with similar results.

Extended Data Fig. 3 Representative plexiform neurofibroma and nerve tissues within the brachial plexus of Nf1flox/flox;PostnCre mice before after treatment with vehicle vs cabozantinib.

a, H&E stained photomicrographs of plexiform neurofibromas and nerve tissues within the brachial plexus of mice of 4 month old Nf1flox/flox;PostnCre mice prior to treatment (top) and after 12 weeks treatment (7 month old) with either vehicle (middle) or cabozantinib (bottom). b, The number of tumors in the brachial plexus was quantified as shown in the bar blot. Bars represent the standard error of the mean. The number of independent mice evaluated in each group were as follows Nf1flox/flox;PostnCre prior to treatment (4 months, n = 5), vehicle treated (7 months, n = 13), cabozantinib treated (7 mo, n = 16). *Adjusted P-value = 0.0254, vehicle vs cabozantinib at 7 months (one-way ANOVA with Tukey’s multiple comparisons test).

Source data

Extended Data Fig. 4 Kinome analysis of murine plexiform neurofibromas after treatment with 1 day and 3 days of cabozantinib.

Volcano plot showing log2- fold change MIB binding (LFQ intensity) after a, 1 day (n = 3) and b, 3 days (n = 5) of cabozantinib vs vehicle (n = 4) control treated sciatic nerve tumor tissue plotted against the −log10 Benjamini-Hochberg adjusted P value of unpaired, two-tailed Student’s t-tests. The dotted line denotes FDR = 0.05. Increased MIB binding was observed of a number of kinases known to modulate an array of cellular processes including proliferation and survival (RPS6KB1), migration and adhesion (PTK2), interferon signaling (JAK1 and TYK2), NF-kappa B signaling (IKBKB), cell cycle and DNA damage response (CDK5 and NEK1), and long-term potentiation and neurotransmitter release (CAMK2A). Kinases with decreased MIB binding after 3 days of cabozantinib treatment including those modulating B lymphocyte development, differentiation and signaling (BTK), cAMP-dependent signaling (PRKACA and PRKACB), and regulation of immune cell chemotaxis and mast cell degranulation (FGR). None of these kinases were cognate targets of cabozantinib and their suppression was transient, likely reflective of an early cellular stress response invoked by cabozantinib.

Source data

Extended Data Fig. 5 Top 20 decreased kinases in MIB competition proteomics assays with cabozantinib.

Stacked bar plot for the top 20 decreased kinases representing the summed log2 fold change in MIB binding for two biological replicates incubated in vitro at varying concentrations of cabozantinib vs DMSO.

Source data

Extended Data Fig. 6 Evaluation of AXL as a cabozantinib target in plexiform neurofibromas of Nf1 mutant mice.

a, MIB binding (LFQ intensity) for AXL in tumor bearing sciatic nerve tissue of Nf1 mutant mice as compared to the WT control. P-value (unpaired, two-tailed Students t-test) <0.0001 as shown. Nerve tissues from n = 3 mice (WT) and n = 4 mice (Nf1flox/flox;PostnCre) were analyzed in one experiment. Data are presented as mean ± SEM. b, AXL was detected by western blot in sciatic nerve tissue lysates from plexiform neurofibroma bearing Nf1flox/flox;PostnCre mice and WT control. GAPDH is shown as the loading control. AXL levels normalized to GAPDH were significantly increased in tumor bearing tissue relative to the WT control, P = 0.05 by two-tailed, Mann-Whitney test. Nerve tissue lysates from n = 3 mice (WT) and n = 4 mice (Nf1flox/flox;PostnCre) were analyzed in two independent experiments. c, AXL expression was detected by immunohistochemistry in WT (PostnCre negative) and Nf1flox/flox;PostnCre tumor bearing tissues following 12 weeks of treatment with either vehicle or cabozantinib. The negative control is shown at the inset. The experiment was conducted two times independently with similar results. d, The percentage of AXL high positive pixels normalized to the tissue area was quantified using ImageJ software and the IHC Profiler plugin. The number of independent mice evaluated per treatment condition were as follows, WT (n = 3), vehicle Nf1flox/flox;PostnCre (n = 6), and cabozantinib Nf1flox/flox;PostnCre (n = 6). Three independent tissue regions were scored from each mouse. The experiment was conducted once. ***Adjusted P-value = 0.0003 vehicle vs cabozantinib, Nf1flox/flox;PostnCre. **Adjusted P-value = 0.0059 WT vs vehicle Nf1flox/flox;PostnCre (one-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean ± SEM. e, AXL, GSK3β, pAKT, and pMEK1/2 and GAPDH were detected by western blot in tumor bearing sciatic nerve tissues from Nf1flox/flox;PostnCre mice treated with either cabozantinib or the vehicle control. The experiment was conducted two times independently with similar results.

Source data

Extended Data Fig. 7 Cabozantinib modulates multiple cellular constituents of plexiform neurofibroma by abrogating AXL signaling.

a, Primary Nf1-/- Schwann cells were stimulated with GAS6 (200 ng/mL) in the presence or absence of cabozantinib (100 nM). Proliferation was assessed by manual cell counting after 48 hours (n = 3 replicates per condition). **Adjusted P-value = 0.0091 unstimulated vs GAS6, **Adjusted P-value =0.0053 GAS6 vs GAS6 + cabozantinib (one-way ANOVA with Sidak’s multiple comparisons test). Data are presented as mean ± SEM. b, pAXL was detected by western blot following stimulation with GAS6 (250 ng/mL) in the presence or absence of cabozantinib (2500 nM). GAPDH is shown as a loading control. The experiment was conducted two times independently with similar results. c, Murine embryonic fibroblasts were stimulated with GAS6 (200 ng/mL) in the presence or absence of cabozantinib (2000 nM). Proliferation was assessed by manual cell counting after 48 hours (n = 6 replicates per condition). **Adjusted P-value = 0.0032 unstimulated vs cabozantinib only, ****Adjusted P-value <0.0001 unstimulated vs GAS6 only, ****Adjusted P-value <0.0001 GAS6 vs GAS6 + cabozantinib (one-way ANOVA with Sidak’s multiple comparisons test). Data are presented as mean ± SEM. d, pAXL was detected by western blot in primary murine embryonic fibroblasts following stimulation with GAS6 (250 ng/mL) from 0 to 30 minutes in the presence or absence of cabozantinib (2000 nM). GAPDH is shown as a loading control. The experiment was conducted two times independently with similar results. e, Human umbilical cord blood derived endothelial colony forming cells (ECFCs) were plated and stimulated with GAS6 (200 ng/mL) in the presence or absence of cabozantinib (100 nM). Proliferation was assessed by manual cell counting after 48 hours (n = 6 replicates per condition). ***Adjusted P-value = 0.0003 unstimulated vs GAS6, *Adjusted P-value = 0.0321 GAS6 vs GAS6 + cabozantinb, ns = not statistically significant, adjusted P-value = 0.0714 (one-way ANOVA with Sidak’s multiple comparisons test). Data are presented as mean ± SEM. pAXL was detected by western blot in ECFCs following stimulation with GAS6 (200 ng/mL) from 0 to 30 minutes in the presences or absence of cabozantinib (1000 nM). GAPDH is shown as a loading control.

Source data

Extended Data Fig. 8 Cabozantinib does not induce apoptosis in Schwann cells or plexiform neurofibroma tumor tissues in vivo.

a, Cabozantinib does not induce apoptosis by caspase 3/7 glo assay in human NF1-/- SC across of range of doses from 39 nM to 5 µM. By contrast, a robust increase in caspase 3/7 activity was observed in NF1-/- Schwann cells treated with Navitoclax (1 µM) as a positive control shown at the right. n = 3 independent cell culture wells were analyzed per condition over two independent experiments. ****Adjusted P-value < 0.0001 Navitoclax vs all other conditions (one-way ANOVA with Tukey’s multiple comparisons test). Data are presented as mean ± SD. b, Representative nerve tissues in cabozantinib (XL184) and vehicle treated mice are negative for TUNEL staining indicating that cabozantinb does not induce apoptosis. Positive and negative controls are shown at the top of the panel. The experiment was conducted two times independently with similar results.

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Extended Data Fig. 9 Increases in sAXL following cabozantinib treatment in Nf1flox/flox;PostnCre mice.

a, Cartoon schematic depicting release of soluble AXL following binding of Gas6 and proteolytic cleavage. b, Soluble AXL was measured by ELISA in the plasma of WT (PostnCre negative) (n = 8) and Nf1flox/flox;PostnCre mice following 12 weeks of treatment with either vehicle (n = 13) or cabozantinib (n = 16). *Adjusted P-value = 0.0167 WT vs cabozantinib Nf1flox/flox;PostnCre, *Adjusted P-value = 0.0179 vehicle vs cabozantinib Nf1flox/flox;PostnCre (one-way ANOVA with Tukey’s multiple comparisons test).

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Extended Data Fig. 10 sAXL levels and proangiogenic CHSPCs vs clinical response to cabozantinib in trial participants.

a, A panel of 45 cytokines including soluble AXL (sAXL) were analyzed in the serum of clinical trial participants (n = 6 with SD, n = 8 with PR). The barplot depicts the change in the mean level of sAXL (ng/mL) between baseline/early (C0/C15) and later cycles (C2/C4) by clinical response of study participants (SD vs PR; P = 0.08 by two-tailed, Mann-Whitney test). Grubb’s test with an alpha value of 0.05 did not identify any statistical outliers within the dataset. Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. b, The frequency of proangiogenic CHSPCs was analyzed in the peripheral blood in n = 13 participants (n = 6 with SD, and n = 7 with PR) collected at 4 time points: prior to cycle 1/baseline (C0), cycle 1 day 15 (C1D15) end of cycle 2 (C2), and end of cycle 4 (C4). The change in the mean frequency of proangiogenic CHSPCs from early (C0/C1D15) to later (C2/C4) treatment cycles was plotted in participants with SD vs PR (p = 0.6282 by two-tailed, Mann Whitney test). Whiskers extend from the minima to maxima. The center line represents the median. The box spans the 25th to 75th percentiles. c, Gating strategy for identification of proangiogenic CHSPCs by flow cytometry.

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Supplementary information

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Reporting Summary

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CHSPC and cytokine data

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Fisher, M.J., Shih, CS., Rhodes, S.D. et al. Cabozantinib for neurofibromatosis type 1–related plexiform neurofibromas: a phase 2 trial. Nat Med 27, 165–173 (2021). https://doi.org/10.1038/s41591-020-01193-6

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