Abstract
The Pediatric Preclinical Testing Program (PPTP) previously reported the activity of the EZH2 inhibitor tazemetostat (EPZ6438) against xenograft models of rhabdoid tumors. Here we determined whether an inhibitor of EZH2 enhanced the effect of standard of care chemotherapeutic agents: irinotecan, vincristine, and cyclophosphamide. EPZ011989 significantly prolonged time to event in all 6 rhabdoid models studied but did not induce tumor regression. The addition EPZ011989 to standard of care agents significantly improved time to event in at least one model for each of the agents studied, although this effect was observed in only a minority of the combination testing experiments.
Keywords: Rhabdomyosarcoma, Xenograft Models, Pediatric Cancer
INTRODUCTION
Previously the Pediatric Preclinical Testing Program evaluated the EZH2 inhibitor tazemetostat (EPZ6438) and observed activity largely restricted to malignant rhabdoid tumor (MRT) models,1 consistent with the suggestion that tumors with deletion of SMARCB1 and hence deficient in the SWI/SNF complex have a synthetic lethal dependency on EZH2.2 In adult patients, tazemetostat is under clinical evaluation for a range of cancers, including SMARCB1 and SMARCA4-deficient cancers (e.g., epithelioid sarcomas). In 2020, tazemetostat received accelerated approval from FDA for the treatment of metastatic or locally advanced epithelioid sarcoma in adult and adolescent patients ≥ 16 years who are not eligible for complete resection. Tazemetostat is also being studied in adults with follicular lymphoma. Pediatric MATCH (NCT03213665) is studying tazemetostat for patients with relapsed or refractory advanced solid tumors, non-Hodgkin lymphoma, or histiocytic disorders with EZH2, SMARCB1, or SMARCA4 gene mutations.
MRTs typically arise during infancy and have poor prognosis.3,4 Treatment consists of surgery, intensive chemo-radiation therapy with or without stem-cell transplant.5 Several studies suggest that regimens developed to treat rhabdomyosarcoma can result in long-term disease free status for children with MRTs.6–9 Here we have evaluated EPZ011989, a potent EZH2 inhibitor with structural properties and preclinical activity very similar to those of tazemetostat,10 both as a single agent and in combination with agents used in the treatment of rhabdomyosarcoma and other sarcomas of childhood.
MATERIALS AND METHODS
In vivo testing:
C.B.17SC scid−/− (C.B-Igh-1b/IcrTac-Prkdcscid) female mice (Taconic, Germantown, NY) were used to propagate subcutaneous sarcoma xenografts. All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care and use committee at UTHSA. G401, KT-12, KT-14 have been described previously.1 RBD1 was from a lung metastasis, and RBD2 from a liver mass (9 month female) that is negative for SMARCB1 by IHC, or mRNA expression. Genomic characterization results are provided in Supplemental Table 1 for all models studied except for RBD-1 and KT-16, and each model with data shows SMARCB1 deep deletion or SMARCB1 mutation.11 Ten mice were used in each control or treatment group. Details of the statistical analytic methods are provided in Appendix 1. Combination testing results were analyzed as previously described,12 with Bonferroni-corrected significance level α = 0.01 due to the five comparisons being made.
Drugs and Formulation:
EPZ011989 was supplied by Epizyme. Drug was formulated in 0.5% sodium carboxymethylcelluose, 0.1% Tween 80, before dosing. Drug was administered at a dose of 250 mg/kg by oral gavage (P.O.) twice daily (BID) for a planned 28 days. Irinotecan was administered at a dose of 2.5 mg/kg (I.P.) days 1–5 to mimic drug exposures in children receiving 50 mg/m2 daily for 5 days, and both vincristine and cyclophosphamide were administered I.P. weekly for 3 consecutive weeks at their maximum tolerated doses: 1 mg/kg and 150 mg/kg, respectively.
RESULTS
In vivo evaluation:
EPZ011989 was tolerated at the dose used although there was considerable weight loss between days 21–28 of dosing, with maximum median weight loss of 16% and with a mortality rate of 10% (6/60). Dosing of EPZ011989 was stopped at day 21 for mice bearing KT-14 xenografts due to >20% weight loss, although mice recovered body weight without mortality. In general, combination therapy was tolerated similarly to single agent EPZ011989. Details of testing results are provided in Supplemental Table 2.
The antitumor activity of the single agents or combinations is summarized in Table 1. Kaplan-Meier analysis is presented in Figure 1 and tumor volume analysis is in Supplemental Figure 1. As a single agent, EPZ011989 was effective at significantly prolonging time to event across all tested models with 3 of 6 models showing markedly prolonged times to event (EFS T/C ratios exceeding 4.0). No tested model treated with single agent EPZ011989 experienced an objective response measure better than PD2, which represents progressive disease with delayed growth (EFS T/C > 2.0). Irinotecan significantly prolonged time to event for all tested models, and one model showed a partial response (PR) and another showed stable disease. Cyclophosphamide significantly prolonged time to event in all six models, with two models showing PR and two models showing SD.
Table 1:
EPZ011989 as single agent and in combination for all PDX models*
| Model | Agent | KM med (days) | EFS T – C (days) | EFS T/C | p-value (vs control) | p-value (combo vs EPZ011989) | p-value (combo vs cytotoxic agent) | minRTV mean + SD | minRTV p-value | Obj. Response |
|---|---|---|---|---|---|---|---|---|---|---|
| G401 | Control | 9.5 | -- | -- | -- | -- | -- | 2.814±0.516 | -- | PD |
| EPZ011989 | 41.7 | 32.1 | 4.37 | p < 0.001 | -- | -- | 1.429±0.586 | p < 0.001 | PD2 | |
| Irinotecan | 26.1 | 16.6 | 2.74 | p < 0.001 | -- | -- | 1.404±0.451 | p < 0.001 | PD2 | |
| EPZ011989 + Irinotecan | 58.2 | 48.6 | 6.10 | p < 0.001 | 0.053 | <0.001 | 0.594±0.412 | p < 0.001 | SD | |
| Vincristine | 37.3 | 27.8 | 3.91 | p < 0.001 | -- | -- | 0.981±0.293 | p < 0.001 | PD2 | |
| EPZ011989 + Vincristine | 39.4 | 29.9 | 4.13 | p < 0.001 | 0.327 | 0.548 | 0.681±0.158 | p < 0.001 | PD2 | |
| Cyclophosphamide | 29.1 | 19.6 | 3.05 | p < 0.001 | -- | -- | 1.703±0.530 | p < 0.001 | PD2 | |
| EPZ011989 + Cyclophosphamide | 67.8 | 58.2 | 7.10 | p < 0.001 | 0.012 | <0.001 | 0.284±0.365 | p < 0.001 | PR | |
| RBD1 | Control | 19.1 | -- | -- | -- | -- | -- | 1.553±0.366 | -- | PD |
| EPZ011989 | 28.2 | 9.1 | 1.48 | p < 0.001 | -- | -- | 1.096±0.252 | p = 0.033 | PD1 | |
| Irinotecan | 45.3 | 26.2 | 2.38 | p < 0.001 | -- | -- | 0.329±0.422 | p < 0.001 | PR | |
| EPZ011989 + Irinotecan | 47.1 | 28.0 | 2.47 | p < 0.001 | <0.001 | 0.142 | 0.067±0.083 | p < 0.001 | PR | |
| Vincristine | > 168 | > 148 | > 8 | p < 0.001 | -- | -- | 0.000±0.000 | p < 0.001 | MCR | |
| EPZ011989 + Vincristine | 104.0 | 84.6 | 5.44 | p < 0.001 | <0.001 | 0.085 | 0.036±0.107 | p < 0.001 | MCR | |
| Cyclophosphamide | 103.0 | 83.4 | 5.38 | p < 0.001 | -- | -- | 0.000±0.000 | p < 0.001 | MCR | |
| EPZ011989 + Cyclophosphamide | 108.0 | 88.7 | 5.65 | p < 0.001 | <0.001 | 0.630 | 0.000±0.000 | p < 0.001 | MCR | |
| RBD2 | Control | 15.0 | -- | -- | -- | -- | -- | 2.285±0.510 | -- | PD |
| EPZ011989 | 19.8 | 4.8 | 1.32 | p = 0.001 | -- | -- | 1.714±0.526 | p = 0.029 | PD1 | |
| Irinotecan | 24.0 | 9.0 | 1.60 | p < 0.001 | -- | -- | 1.057±0.481 | p < 0.001 | PD1 | |
| EPZ011989 + Irinotecan | 47.6 | 32.7 | 3.18 | p < 0.001 | 0.002 | 0.007 | 0.715±0.300 | p < 0.001 | PD2 | |
| Vincristine | 44.5 | 29.5 | 2.97 | p < 0.001 | -- | -- | 0.904±0.479 | p < 0.001 | PD2 | |
| EPZ011989 + Vincristine | 67.5 | 52.5 | 4.51 | p < 0.001 | 0.001 | 0.043 | 1.016±0.582 | p < 0.001 | PD2 | |
| Cyclophosphamide | 32.8 | 17.8 | 2.19 | p = 0.013 | -- | -- | 1.344±0.443 | p < 0.001 | PD2 | |
| EPZ011989 + Cyclophosphamide | 31.0 | 16.0 | 2.07 | p < 0.001 | 0.055 | 0.940 | 1.475±0.260 | p < 0.001 | PD1 | |
| KT-12 | Control | 15.9 | -- | -- | -- | -- | -- | 2.719±1.221 | -- | PD |
| EPZ011989 | 73.4 | 57.5 | 4.62 | p < 0.001 | -- | -- | 0.786±0.398 | p < 0.001 | PD2 | |
| Irinotecan | 44.8 | 28.9 | 2.82 | p < 0.001 | -- | -- | 0.716±0.485 | p < 0.001 | PD2 | |
| EPZ011989 + Irinotecan | 60.2 | 44.3 | 3.79 | p < 0.001 | 0.285 | 0.033 | 0.562±0.473 | p < 0.001 | SD | |
| Vincristine | 63.6 | 47.7 | 4.01 | p < 0.001 | -- | -- | 0.563±0.312 | p < 0.001 | PR | |
| EPZ011989 + Vincristine | 98.4 | 82.6 | 6.20 | p < 0.001 | 0.007 | 0.004 | 0.140±0.153 | p < 0.001 | CR | |
| Cyclophosphamide | 64.3 | 48.4 | 4.05 | p < 0.001 | -- | -- | 0.521±0.344 | p < 0.001 | PD2 | |
| EPZ011989 + Cyclophosphamide | 80.8 | 64.9 | 5.09 | p < 0.001 | 0.416 | 0.207 | 0.342±0.346 | p < 0.001 | PR | |
| KT-14 | Control | 25.6 | -- | -- | -- | -- | -- | 2.222±0.936 | -- | PD |
| EPZ011989 | 34.8 | 9.2 | 1.36 | p < 0.001 | -- | -- | 1.525±0.664 | p = 0.139 | PD1 | |
| Irinotecan | 40.7 | 15.1 | 1.59 | p < 0.001 | -- | -- | 0.911±0.439 | p < 0.001 | PD1 | |
| EPZ011989 + Irinotecan | 117.0 | 91.5 | 4.57 | p < 0.001 | 0.068 | <0.001 | 0.729±0.265 | p < 0.001 | PD2 | |
| Vincristine | 45.9 | 20.3 | 1.79 | p < 0.001 | -- | -- | 0.926±0.373 | p < 0.001 | PD1 | |
| EPZ011989 + Vincristine | > 125 | > 99.4 | > 4.88 | p < 0.001 | 0.004 | <0.001 | 0.392±0.132 | p < 0.001 | PR | |
| Cyclophosphamide | 93.3 | 67.7 | 3.64 | p < 0.001 | -- | -- | 0.681±0.321 | p < 0.001 | PD2 | |
| EPZ011989 + Cyclophosphamide | 102.0 | 76.3 | 3.98 | p < 0.001 | 0.143 | 0.538 | 0.730±0.172 | p < 0.001 | PD2 | |
| KT-16 | Control | 13.1 | -- | -- | -- | -- | -- | 2.288±0.385 | -- | PD |
| EPZ011989 | 72.2 | 59.1 | 5.51 | p < 0.001 | -- | -- | 0.910±0.271 | p < 0.001 | PD2 | |
| Irinotecan | 82.3 | 69.2 | 6.29 | p < 0.001 | -- | -- | 0.548±0.364 | p < 0.001 | SD | |
| EPZ011989 + Irinotecan | 147.0 | 134.2 | 11.26 | p < 0.001 | 0.009 | 0.066 | 0.238±0.130 | p < 0.001 | PR |
FIGURE 1.
Event-free survival in response to EPZ011989 as a single agent and in combination across all PDX models
For the 6 models tested with the combination of irinotecan and EPZ011989, the combination was significantly superior to single agent irinotecan for three lines (G401, RBD2, and KT-14). For these three lines the objective response measure improved for each (PD2 to SD, PD1 to PD2, and PD1 to PD2, respectively). For KT-16, the objective response measure also improved from SD to PR.
For the 5 models tested with the combination of vincristine and EPZ011989, the combination was significantly superior to single agent vincristine (p< 0.01) for two lines (KT-12 and KT-14). KT-12 and KT-14 also showed improvement in their objective response measure for the combination compared to single agent vincristine (PR to CR and PD1 to PR, respectively).
For the 5 models tested with the combination of cyclophosphamide and EPZ011989, the combination was significantly superior to single agent cyclophosphamide for a single line (G401). The objective response measure improved for the combination versus single agent cyclophosphamide for G401 and KT-12 (both PD2 to PR). The objective response measure was lowered for RBD2 (PD2 to PD1).
Therapeutic Enhancement, in which the activity of the combination is significantly superior to that of either single agent, was identified in only three settings. These were for irinotecan against RBD2 and for vincristine in the KT-12 and KT-14 models.
DISCUSSION
Malignant rhabdoid tumors are aggressive cancers that arise in brain or in soft tissues. MRT is associated with relatively short survival, although several reports indicate that regimens developed for treatment of rhabdomyosarcoma may extend disease-free survival in some patients. In prior testing by the PPTP, tazemetostat showed significant prolongation in time to event against several MRT models, and this agent is in clinical trials for patients at relapse with loss of SMARCB1. One future direction for tazemetostat clinical development could be to combine an EZH2 inhibitor with agents used to treat rhabdomyosarcoma. We were therefore interested in whether combination of an EZH2 inhibitor would enhance the activity of cytotoxic agents used in current high-risk rhabdomyosarcoma protocols, specifically, vincristine, cyclophosphamide and irinotecan. Although pharmacodynamic testing was not performed, the EPZ011989 dose and schedule utilized have been shown to markedly reduce H3K27me3 levels following seven days of treatment.10
EPZ011989 was effective at increasing EFS in all models but did not cause tumor regressions as a single agent. A key question for an investigational agent is whether its addition to standard of care agents can increase efficacy compared to the standard of care agent alone. For EPZ011989, our results demonstrate that its addition significantly improved time to event in 3 of 6 models for irinotecan, 2 of 5 models for vincristine, and 1 of 5 models for cyclophosphamide. The ability of EPZ011989 to increase the efficacy of standard of care agents occurred both for models for which EPZ011989 showed its greatest single agent effect (G401, KT-12, and KT-16 with EFS T/C > 4.0) as well as for models with a smaller single agent effect (RBD2 and KT-14 with EFS T/C < 1.4).
In summary, EPZ011989 had moderate single agent in vivo activity against our panel of MRT xenografts as demonstrated by varying degrees of extension in time to event. Its addition to standard of care agents significantly improved time to event in at least one model for each of the agents studied, although this effect was observed in a minority of the combination testing experiments.
Supplementary Material
Supplemental Table 2: Detailed Testing Results
ACKNOWLEDGEMENTS
We thank Abhik Bandyophadhyay, Vanessa Del Pozo, Samson Ghilu, and Edward Favours for technical assistance, and we thank Epizyme for provision of EPZ011989. This work was supported by NCI grants: UO1 CA199297, U01 CA199222, and P30 CA054174.
Abbreviation
- PPTP
Pediatric Preclinical Testing Program
- MRT
Malignant rhabdoid tumor
- ATRT
Atypical teratoid rhabdoid tumor
- RMS
Rhabdomyosarcoma
Footnotes
Conflict of Interest: The authors have no conflicts.
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
REFERENCES
- 1.Kurmasheva RT, Sammons M, Favours E, et al. Initial testing (stage 1) of tazemetostat (EPZ-6438), a novel EZH2 inhibitor, by the Pediatric Preclinical Testing Program. Pediatr Blood Cancer. 2017;64(3). [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Wilson BG, Wang X, Shen X, et al. Epigenetic antagonism between polycomb and SWI/SNF complexes during oncogenic transformation. Cancer cell. 2010;18(4):316–328. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Burger PC, Yu IT, Tihan T, et al. Atypical teratoid/rhabdoid tumor of the central nervous system: a highly malignant tumor of infancy and childhood frequently mistaken for medulloblastoma: a Pediatric Oncology Group study. Am J Surg Pathol. 1998;22(9):1083–1092. [DOI] [PubMed] [Google Scholar]
- 4.Packer RJ, Biegel JA, Blaney S, et al. Atypical teratoid/rhabdoid tumor of the central nervous system: report on workshop. J Pediatr Hematol Oncol. 2002;24(5):337–342. [DOI] [PubMed] [Google Scholar]
- 5.Rorke LB, Packer R, Biegel J. Central nervous system atypical teratoid/rhabdoid tumors of infancy and childhood. J Neurooncol. 1995;24(1):21–28. [DOI] [PubMed] [Google Scholar]
- 6.Olson TA, Bayar E, Kosnik E, et al. Successful treatment of disseminated central nervous system malignant rhabdoid tumor. J Pediatr Hematol Oncol. 1995;17(1):71–75. [DOI] [PubMed] [Google Scholar]
- 7.Weinblatt M, Kochen J. Rhabdoid tumor of the central nervous system. Med Pediatr Oncol. 1992;20(3):258. [DOI] [PubMed] [Google Scholar]
- 8.Zimmerman MA, Goumnerova LC, Proctor M, et al. Continuous remission of newly diagnosed and relapsed central nervous system atypical teratoid/rhabdoid tumor. J Neurooncol. 2005;72(1):77–84. [DOI] [PubMed] [Google Scholar]
- 9.Chi SN, Zimmerman MA, Yao X, et al. Intensive multimodality treatment for children with newly diagnosed CNS atypical teratoid rhabdoid tumor. J Clin Oncol. 2009;27(3):385–389. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Campbell JE, Kuntz KW, Knutson SK, et al. EPZ011989, A Potent, Orally-Available EZH2 Inhibitor with Robust in Vivo Activity. ACS medicinal chemistry letters. 2015;6(5):491–495. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Rokita JL, Rathi KS, Cardenas MF, et al. Genomic Profiling of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design. Cell Rep. 2019;29(6):1675–1689 e1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Houghton PJ, Morton CL, Gorlick R, et al. Stage 2 combination testing of rapamycin with cytotoxic agents by the Pediatric Preclinical Testing Program. Molecular cancer therapeutics. 2010;9(1):101–112. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Supplemental Table 2: Detailed Testing Results