Evaluation of VTP-50469, a menin-MLL1 inhibitor, against Ewing sarcoma xenograft models by the pediatric preclinical testing consortium
Raushan T. Kurmasheva1
Vanessa Del Pozo1
Doris A. Phelps1
Gerard M. McGeehan2
Stephen W. Erickson3
Malcolm A. Smith4
Peter J. Houghton1
Background: VTP-50469 is a potent inhibitor of the menin-MLL1 interaction and is implicated in signaling downstream of EWSR1-FLI1.
Procedure: VTP-50469 was evaluated against seven Ewing sarcoma (EwS) xenograft models and in vitro against EwS cell lines.
Results: VTP-50469 showed limited antitumor activity, statistically significantly slowing tumor progression in four tumor models but with no evidence of tumor regression. In vitro, the IC50 con- centration was 10 nM for the mixed lineage leukemia (MLL)-rearranged leukemia cell line MV4;11, but > 3 M for EwS cell lines.
Conclusions: In contrast to its high level of activity against MLL1-rearranged leukemia xenografts, VTP-50469 shows little activity against EwS models.
Ewing sarcoma, pediatric oncology, xenograft
1 UT Health San Antonio, Greehey Children’s
Cancer Research Institute,, San Antonio, Texas
2 Syndax Pharmaceuticals, Waltham,
3 Research Triangle Park, Research Triangle
Institute,, North Carolina
4 NCI, Cancer Therapy Evaluation Program,,
Correspondence Raushan T. Kurmasheva, Greehey Children’s Cancer Research Institute, 8403 Floyd Curl Drive, San Antonio, TX 78229.
Email: [email protected]
Funding information National Cancer Institute, Grant/Award Num- bers: 1U01CA199222-01, P30 CA054174, UO1 CA199297
MLL1, the mammalian homolog of drosophila Trithorax (trx), posi- tively regulates HOX gene expression during development.1 The activ- ity of trx group proteins is balanced by the repressive activity of Polycomb group (PcG) genes.2 MLL1 (KMT2A), a lysine methyltrans- ferase, binds promoters of HOX genes resulting in H3 Lys 4 methyla- tion and H3 and H4 acetylation.3 Dysregulation of HOX genes occurs in several cancers, suggesting a critical role for developmental pro- grams in transformation.4-6 Although the role of MLL1 translocations is well established for several leukemias,1 less is known regarding the role of MLL1 in solid tumors. In leukemias, the oncogenic activ- ity of MLL1 fusion proteins is dependent on association with menin, a scaffolding protein that binds MLL1 and MLL4 (KMT2B) in the con- text of TrxG COMPASS complexes.7 Consequently, small molecules
Abbreviations: EwS, Ewing sarcoma; MLL, mixed lineage leukemia; PcG, polycomb group; PPTC, Pediatric Preclinical Testing Consortium; RTV, Relative tumor volumes.
that inhibit the Menin-MLL1 interaction have potential therapeutic value for treatment of MLL1-rearranged leukemia.8 Posterior HOXD genes are overexpressed in Ewing sarcoma (EwS).9 Promoter regions for these genes are characterized by mixed lineage leukemia (MLL)- mediated H3K4me3 marks and are devoid of recessive H3K27me3 marks.9 Recently, evidence has been presented that the tumorigenicity of EwS cells is dependent on the Menin-MLL1 interaction.10 Here we have evaluated VTP-50469, a potent inhibitor of Menin-MLL1 interac- tions that shows profound activity against Pediatric Preclinical Testing Consortium (PPTC) MLL-rearranged leukemia models.11
⦁ MATERIALS AND METHODS
⦁ In vivo testing
C.B.17SC scid−/− (C.B-Igh-1b/IcrTac-Prkdcscid) female mice were main- tained under barrier conditions and experiments were conducted
Pediatr Blood Cancer. 2020;e28284. https://doi.org/10.1002/pbc.28284
○c 2020 Wiley Periodicals, Inc.
TA B L E 1 Summary of antitumor activity of VTP-50469 against EwS xenograft models
Treatment KM median (days) EFS T-C
EFS T/C P value
Gehan-Wilcoxon Median response Minimum relative tumor volume
CHLA258 Control 16.2 1.971 ± 0.771
VTP-50469 20.1 3.9 1.24 0.036 PD1 1.553 ± 0.430
ES-1 Control 10.4 3.047 ± 0.816
VTP-50469 14.0 3.6 1.35 0.046 PD1 2.612 ± 0.991
ES-4 Control 10.7 2.494 ± 0.616
VTP-50469 12.2 1.5 1.14 0.186 PD1 2.092 ± 0.460
ES-6 Control 12.4 2.066 ± 0.280
VTP-50469 11.5 –0.9 0.93 0.084 PD1 2.333 ± 0.953
EW-5 Control 13.1 1.709 ± 0.370
VTP-50469 20.8 7.8 1.6 <0.001 PD1 1.241 ± 0.394
EW-8 Control 6.1 5.014 ± 0.931
VTP-50469 10.6 4.5 1.74 0.015 PD1 3.497 ± 1.177
NCH-EWS-1 Control 10.3 2.741 ± 1.078
VTP-50469 10.4 0.1 1.01 0.665 PD1 3.112 ± 0.823
using protocols and conditions approved by The Institutional Ani- mal Care and Use Committee at UTHSCSA as previously described.12 Details of the statistical analytic methods are provided in Appendix 1.
⦁ Drugs and formulation
VTP-50469 was provided to the PPTC by Syndax Pharmaceuticals Inc., through the Cancer Therapy Evaluation Program (NCI). VTP-50469 was suspended in the required amount of vehicle (0.5% natrosol + 1% polysorbate-80). The resulting formulation was sonicated in a water bath set to 37◦C until completely dissolved or visually uniform sus- pensions were achieved. Formulated drug was stored at 4◦C for up to one month. VTP-50469 was administered by oral gavage (p.o.) at 120 mg/kg, twice daily (b.i.d.) for a planned 28 consecutive days.
⦁ In vitro testing
The potency of VTP-50469 was evaluated against four EwS cell lines (ES-1, ES-4, ES-6, and EW-8). Each line had the EWSR1-FLI1 type 1 translocation. VTP-50469 was also tested against the MV4;11 cell line that had the MLL-AF4 (KMT2A-AFF1) fusion. Cells were exposed to VTP-50469 for 96 hours, and viability was assessed by Alamar Blue staining, as described previously.13
For CHLA-258 and EW-8 cell lines, the VTP-50469 dose was reduced to 100 mg/kg b.i.d. because of excessive toxicity observed in mod- els treated at 120 mg/kg b.i.d. For the five EwS models treated at 120 mg/kg b.i.d., excessive mortality (18 of 50) was observed in the absence of antecedent weight loss. For the remaining two models (CHLA-258 and EW-8) treated at 100 mg/kg b.i.d., 1 of 20 mice in the treatment group died (5%). VTP-50469 caused a statistically sig-
nificant growth delay in four EwS models (Table 1). Among models with significant slowing of tumor growth, the ratio of median time to event for the treated versus control groups (EFS T/C) ranged from
1.24 to 1.74. There were no tumor regressions, and the mean min- imum relative tumor volumes (RTV) for treated groups ranged from
1.2 to 3.5 (Table 1). The overall objective response classification for all models was progressive disease 1 (PD1). Kaplan-Meier event-free survival (EFS) for each tumor line is shown in Figure 1. The expression (mRNA) and mutation status of KMT2A and Menin (MEN1) in the PPTC leukemia and solid tumor models is shown in Supporting Information Figures S1 and S2.
Because VTP-50469 demonstrated limited activity against EwS xenograft models at a dose and schedule that was highly active against MLL-rearranged leukemias, we determined the sensitivity of EwS cell lines in comparison with the MLL-rearranged leukemia cell line MV4;11. Cells were exposed to VTP-50469 at concentrations from 3 nM to 3 M for 96 hours. The MV4;11 cell line was very sensitive
to VTP-50469 (IC50 = 10 nM), whereas for all EwS cell lines the IC50
was > 3 M (Figure 2).
MLL translocations occur in 5% to 10% of B-cell acute lymphoblas- tic leukemia (ALL) with a similar frequency observed for acute myeloid leukemia (AML). Rearrangements are more frequent in infant leukemias (∼70% of infant ALL). Menin inhibitors are highly active in preclinical models of MLL-rearranged ALL,8,11,14 and clinical trials for this class of agents have been initiated (SNDX-5613 in NCT04065399 and KO-539 in NCT04067336).
Recent work has suggested that Menin-MLL1 interactions may be important for tumorigenesis induced by the EWSR1-FLI1 fusion oncogene in EwS and that this effect may be in part mediated through effects on serine biosynthesis.10,15 MI-503, a small molecule that
FIGURE 1 Kaplan–Meier plots showing probability of being event-free with time. Red line, controls; blue line, VTP-50469 treatment
FIGURE 2 Sensitivity of EwS cell lines compared with MV4;11, a cell line that has the t(4;11)(q21;q23) chromosomal translocation leading to fusion of the MLL gene located on 11q23 and AF4 located on 4q21 (MLL/AF4; or KMT2A/AFF1). Cells were exposed to
VTP-50469 for 96 hours and viability assessed by Alamar Blue staining
disrupts the Menin-MLL1 interaction,8 reduced proliferation and sup- pressed anchorage-independent growth of EwS cell lines.10 In vitro, MI-503 induced loss of both MLL1 and menin, and it also reduced the tumorigenicity of EwS cells that were pretreated with MI-503 prior to inoculation into athymic nude mice.10 mRNA expression in EwS models for KMT2A and Menin (MEN1) shows that expression of KMT2A is lower than in MLL models that have fusions. The only EwS model to have a mutation (missense; E2419K) is SK-NEP-1, a model not used in the current study. Expression of MEN1 was similar in leukemias and EwS models, and no mutations in the EwS models were detected (https://pedcbioportal.org/study?id=pptc#summary).16
VTP-50469, like MI-503, potently disrupts the Menin-MLL1 interaction and is highly active over a broad range of doses in infant leukemias that have MLL1 translocations. Against EwS xenografts, VTP-50469 showed little antitumor activity, statistically significantly slowing tumor growth in several models. However, extension of EFS in these models was modest, and tumor regression was not observed.
Consistent with the in vivo results, EwS cell lines were > 300-fold less sensitive to VTP-50469 than the MLL leukemia cell line MV4;11. Phar- macodynamic studies were not undertaken to determine target inhibi- tion, as VTP-50469 administered at these and lower dose levels on the same schedule has robust activity in several infant leukemia models with MLL1 translocations. Our results are consistent with those of a recent report that found that while MI-503 had in vitro activity against a range of leukemia and solid tumor cell lines, the more selective menin inhibitor BAY-155 was primarily active in AML and ALL models.17
Our results and an examination of the existing literature for menin inhibitors suggest that EwS cells are less dependent on the Menin- MLL1 interaction for survival in comparison with MLL-rearranged leukemias. First, the IC50 values for EwS cell lines are approximately one log greater than those for MLL-rearranged leukemia cell lines for the menin inhibitor MI-503.8,10 Similarly, our in vitro results show much greater sensitivity for a MLL-rearranged leukemia cell line to VTP-50469 in comparison with that observed for EwS cell lines. Sec- ond, the effect of menin inhibitors on the expression of the MLL1 gene fusion target genes (e.g., Hox9 and Meis1) is much greater than the effect of menin inhibition on expression of HoxD genes in EwS cell lines.8,10 Finally, VTP-50469 shows remarkably high in vivo activity against MLL-rearranged leukemia xenograft lines, but shows minimal levels of in vivo activity against EwS models.
CONFLICTS OF INTEREST
Dr. McGeehan is an employee of Syndax Pharmaceuticals. The other authors have no conflicts.
This work was supported by NCI grants UO1 CA199297, U01 CA199222, and P30 CA054174.
Raushan T. Kurmasheva https://orcid.org/0000-0003-3212-2363
⦁ HessJL. Mechanisms of transformation by MLL. Crit Rev Eukaryot Gene Expr. 2004;14(4):235-254.
⦁ KassisJA, KennisonJA, TamkunJW. Polycomb and trithorax group genes in drosophila. Genetics. 2017;206(4):1699-1725.
⦁ NakamuraT, MoriT, TadaS, et al. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell. 2002;10(5):1119-1128.
⦁ GalloM, HoJ, CoutinhoFJ, et al. A tumorigenic MLL-homeobox net- work in human glioblastoma stem cells. Cancer Res. 2013;73(1):417- 427.
⦁ MunteanAG, HessJL. The pathogenesis of mixed-lineage leukemia.
Annu Rev Pathol. 2012;7:283-301.
⦁ XuB, LiSH, ZhengR, et al. Menin promotes hepatocellular carcinogen- esis and epigenetically up-regulates Yap1 transcription. Proc Natl Acad Sci U S A. 2013;110(43):17480-17485.
⦁ YokoyamaA, SomervailleTC, SmithKS, Rozenblatt-RosenO, Meyer- sonM, ClearyML. The menin tumor suppressor protein is an essen- tial oncogenic cofactor for MLL-associated leukemogenesis. Cell. 2005;123(2):207-218.
⦁ BorkinD, HeS, MiaoH, et al. Pharmacologic inhibition of the Menin- MLL interaction blocks progression of MLL leukemia in vivo. Cancer Cell. 2015;27(4):589-602.
⦁ How to cite this article: KurmashevaRT, BandyopadhyayA, FavoursE, et al. Evaluation of VTP-50469, a menin-MLL1 inhibitor, against Ewing sarcoma xenograft models by the pediatric preclinical testing consortium. Pediatr Blood Cancer. 2020;e28284. https://doi.org/10.1002/pbc.28284
⦁ SvobodaLK, HarrisA, BaileyNJ, et al. Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epige- netic regulation of developmental transcription programs. Epigenetics. 2014;9(12):1613-1625.
⦁ SvobodaLK, BaileyN, Van NoordRA, et al. Tumorigenicity of Ewing sar- coma is critically dependent on the trithorax proteins MLL1 and menin. Oncotarget. 2017;8(1):458-471.
⦁ KrivtsovAV, EvansK, GadreyJY, et al. A Menin-MLL Inhibitor induces specific chromatin changes and eradicates disease in models of mll- rearranged leukemia. Cancer Cell. 2019;36(6):660-673 e611.
⦁ HoughtonPJ, MortonCL, TuckerC, et al. The pediatric preclinical test- ing program: description of models and early testing results. Pediatr Blood Cancer. 2007;49:928-940.
⦁ BidHK, PhelpsDA, XaioL, et al. The bromodomain BET inhibitor JQ1 suppresses tumor angiogenesis in models of childhood sarcoma. Mol Cancer Ther. 2016;15(5):1018-1028.
⦁ LockRB, EvansK, PrichardT, et al. Pediatric Preclinical Testing Consor- tium evaluation of the menin inhibitor, VTP-50469, against xenograft models of MLL-rearranged infant acute lymphoblastic leukemia. Proc Am Assoc Cancer Research. 2018. Abstract 3187.
⦁ SvobodaLK, TehSSK, SudS, et al. Menin regulates the serine biosyn- thetic pathway in Ewing sarcoma. J Pathol. 2018; 245(3): 324-336.
⦁ RokitaJL, RathiKS, CardenasMF, 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.
⦁ BrzezinkaK, NevedomskayaE, LescheR, et al. Characterization of the Menin-MLL interaction as therapeutic cancer target. Cancers. 2020;12(1):E201.VTP50469