Contributions
Abstract: EP382
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The chromosomal translocation t(5;11)(q35;q15.5) encoding the NUP98/NSD1 fusion gene is a frequent rearrangement in pediatric high-risk AML. In more than 80% of all cases, NUP98/NSD1 co-occurs with FLT3-ITD or WT1 mutations. This AML subtype is characterized by a HOXA/B programme, where MLL binds to the N-terminus of the NUP98/NSD1 fusion protein and recruits it to the HOXA and MEIS1 loci. In general, transcriptional activation by MLL depends on its interaction with its cofactor Menin. SNDX-5613 is a novel selective Menin-MLL inhibitor that resides with high affinity in the binding pocket of Menin and disrupts the Menin-MLL interaction. This inhibitor has recently been shown to have substantial single agent activity against MLL-rearranged and NPM1 mutant leukemia. Based on these combined findings, we hypothesized that the NUP98/NSD1 leukaemic programme depends on a functional Menin-MLL interaction that can be targeted by SNDX-5613.
Aims
The aim of this study was to examine the significance of the MLL – MEN1 interaction for NUP98/NSD1-driven gene expression and to explore the potency of SNDX-5613 to interfere with t(5;11)-AML propagation.
Methods
Patient AML samples positive for NUP98/NSD1 were provided by the Princess Maxima Center for Pediatric Oncology. All samples were also positive for FLT3-ITD with VAFs > 0.35. AML samples were co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and treated with different concentrations of SNDX-5613 (Syndax Pharmaceuticals) followed by evaluation of proliferation, gene and protein expression, clonogenicity and differentiation. Changes in cell surface marker expression were investigated after 2 weeks using flow cytometry.
Results
Co-culture of NUP98/NSD1-positive patient samples resulted in a 3 – 6-fold expansion of total cells and a 15 – 200-fold expansion of immature CD34+ CD38- CD117+ CD45RA+ leukaemic cells over a period of 14 days. Treatment of four different NUP98/NSD1-positive AML samples in this setting resulted in a profound dose-dependent inhibition of proliferation with latency of 10 days and IC50 values between 2 and 50 nM. On the contrary, primary AML cells lacking NUP98/NSD1 translocation were not affected even by 250 nM of SNDX-5613, demonstrating on-target mechanism of action of this compound. This growth inhibitory effect was associated with a 70% reduction in clonogenicity both by colony number and size when compared with DMSO-treated cells. Furthermore, SNDX-5613 treatment induced myeloid differentiation in AML blasts as demonstrated by diminished expression of CD34 and CD45RA and concomitant elevated expression of CD11b, CD14 and MNDA. Transcriptional profiling upon SNDX-5613 treatment revealed specific changes in leukemogenic gene expression with downregulation of HOXA7 – 10 and MEIS1. SNDX-5613 also reduced CDK6 levels, a key regulator of cell cycle progression and an established direct transcriptional target of MLL and NUP98/NSD1. Notably, we found that and its potential target gene FLT3 are uniformly downregulated in a dose dependent manner suggesting an impact of this drug on a secondary leukaemic driver.
Conclusion
This study demonstrates the relevance of the Menin-MLL interaction for NUP98/NSD1-driven leukemogenesis in primary patient cells and highlights the pharmacologic inhibition of this interaction as a promising therapeutic option for this poor prognosis AML.
Keyword(s): Flt3-ITD, Fusion, MLL, NUP98
Abstract: EP382
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The chromosomal translocation t(5;11)(q35;q15.5) encoding the NUP98/NSD1 fusion gene is a frequent rearrangement in pediatric high-risk AML. In more than 80% of all cases, NUP98/NSD1 co-occurs with FLT3-ITD or WT1 mutations. This AML subtype is characterized by a HOXA/B programme, where MLL binds to the N-terminus of the NUP98/NSD1 fusion protein and recruits it to the HOXA and MEIS1 loci. In general, transcriptional activation by MLL depends on its interaction with its cofactor Menin. SNDX-5613 is a novel selective Menin-MLL inhibitor that resides with high affinity in the binding pocket of Menin and disrupts the Menin-MLL interaction. This inhibitor has recently been shown to have substantial single agent activity against MLL-rearranged and NPM1 mutant leukemia. Based on these combined findings, we hypothesized that the NUP98/NSD1 leukaemic programme depends on a functional Menin-MLL interaction that can be targeted by SNDX-5613.
Aims
The aim of this study was to examine the significance of the MLL – MEN1 interaction for NUP98/NSD1-driven gene expression and to explore the potency of SNDX-5613 to interfere with t(5;11)-AML propagation.
Methods
Patient AML samples positive for NUP98/NSD1 were provided by the Princess Maxima Center for Pediatric Oncology. All samples were also positive for FLT3-ITD with VAFs > 0.35. AML samples were co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and treated with different concentrations of SNDX-5613 (Syndax Pharmaceuticals) followed by evaluation of proliferation, gene and protein expression, clonogenicity and differentiation. Changes in cell surface marker expression were investigated after 2 weeks using flow cytometry.
Results
Co-culture of NUP98/NSD1-positive patient samples resulted in a 3 – 6-fold expansion of total cells and a 15 – 200-fold expansion of immature CD34+ CD38- CD117+ CD45RA+ leukaemic cells over a period of 14 days. Treatment of four different NUP98/NSD1-positive AML samples in this setting resulted in a profound dose-dependent inhibition of proliferation with latency of 10 days and IC50 values between 2 and 50 nM. On the contrary, primary AML cells lacking NUP98/NSD1 translocation were not affected even by 250 nM of SNDX-5613, demonstrating on-target mechanism of action of this compound. This growth inhibitory effect was associated with a 70% reduction in clonogenicity both by colony number and size when compared with DMSO-treated cells. Furthermore, SNDX-5613 treatment induced myeloid differentiation in AML blasts as demonstrated by diminished expression of CD34 and CD45RA and concomitant elevated expression of CD11b, CD14 and MNDA. Transcriptional profiling upon SNDX-5613 treatment revealed specific changes in leukemogenic gene expression with downregulation of HOXA7 – 10 and MEIS1. SNDX-5613 also reduced CDK6 levels, a key regulator of cell cycle progression and an established direct transcriptional target of MLL and NUP98/NSD1. Notably, we found that and its potential target gene FLT3 are uniformly downregulated in a dose dependent manner suggesting an impact of this drug on a secondary leukaemic driver.
Conclusion
This study demonstrates the relevance of the Menin-MLL interaction for NUP98/NSD1-driven leukemogenesis in primary patient cells and highlights the pharmacologic inhibition of this interaction as a promising therapeutic option for this poor prognosis AML.
Keyword(s): Flt3-ITD, Fusion, MLL, NUP98