Autoren:	Dzama, Margarita
Titel:	Combined targeting of the Menin-MLL chromatin regulatory complex and the FLT3 tyrosine kinase as a novel therapeutic approach against NPM1mut and MLL-r leukemia with concurrent FLT3 mutation
Online-Publikationsdatum:	21-Mai-2021
Erscheinungsdatum:	2021
Sprache des Dokuments:	Englisch
Zusammenfassung/Abstract:	Acute myeloid leukemia (AML) is a heterogeneous clonal disorder characterized by aberrant proliferation and blocked differentiation of immature myeloid cells eventually causing bone marrow failure. NPM1 mutant (NPM1mut) and MLL-rearranged (MLL-r) acute myeloid leukemia subtypes, characterized by aberrant expression of HOX and MEIS1 transcription factors, commonly harbor concurrent activating mutations in the FMS-like tyrosine kinase receptor (FLT3). It has been previously demonstrated that the interaction of Menin and MLL (MLL1, KMT2A) proteins represents a dependency in NPM1mut and MLL-r AML, and therefore a potential therapeutic opportunity against these leukemias. The aim of this study was to investigate the effect of pharmacological inhibition of Menin-MLL complex on FLT3 expression and FLT3 mutant signaling as well as to assess the therapeutic potential of combined Menin-MLL and FLT3 targeting. In order to explore transcriptional changes associated with disruption of the Menin-MLL complex, RNA-sequencing (RNA-seq) of NPM1mut OCI-AML3 and MLL-r MOLM13 and MV411 leukemia cells following inhibition with the small-inhibitory molecule MI-503 was performed. MEIS1 and its target gene FLT3 were found among the most significantly downregulated genes. I confirmed these results in various human NPM1mut or MLL-r cell lines and in primary murine Npm1mut/+Flt3mut/+ leukemia cells. Moreover, I found a profound suppression of the FLT3 mutant allele following MI-503 treatment in the MLL-r MV411 and MOLM13 cells using specific primers followed by quantitative PCR (qPCR). I also observed a significant decrease in FLT3 protein expression upon MI-503 treatment in all tested NPM1mut and MLL-r AMLs as assessed by FACS. Using chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) I could demonstrate that inhibition of Menin and MLL interaction significantly reduces the binding of both proteins to the MEIS1 gene. Next, I evaluated the therapeutic potential of combined Menin-MLL and FLT3 inhibition in the FLT3-ITD positive MLL-r and NPM1mut leukemias. I found synergistic anti-proliferative activity accompanied by enhanced apoptosis upon combined treatment with MI-503 and the specific FLT3 inhibitor Quizartinib compared to single-drug treatment or vehicle control in human MV411 and MOLM13 cells. For murine Npm1mut/+Flt3mut/+ leukemia cells, I used the multi-tyrosine kinase inhibitor Ponatinib as a combination partner for MI-503 in these cells as these cells harbor the F692L gatekeeper mutation that conveys drug resistance to most FLT3 inhibitors, including Quizartinib. Again, similar synergistic suppression of proliferation and colony formation as well as significantly enhanced apoptosis were observed. Similar results were obtained when MI-503 was combined with another FLT3 inhibitor - Gilteritinib, which was recently approved by U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). Human HL-60 and NB4 cells, or the murine Hoxa9-Meis1-transformed cells that served as negative controls (all wildtype for NPM1, MLL, FLT3) were not affected by single drug or combinatorial treatment. To investigate the mechanism of drug synergy I then evaluated the level of phosphorylated (activated) FLT3 (pFLT3) by immunoblotting and flow cytometry. As expected, I observed reduced pFLT3 upon direct FLT3 inhibition and found decreased total FLT3 and pFLT3 with MI-503 monotreatment. The most pronounced pFLT3 reduction was observed with combined Menin-MLL and FLT3 inhibition, most likely reflecting the joint effect of direct pFLT3 inhibition with Quizartinib or Ponatinib treatment and transcriptional suppression of total FLT3 with MI-503 treatment. The observed anti-leukemic effects were confirmed by combining a recently developed second-generation Menin-MLL inhibitor, VTP-50469, that is under first clinical investigation (NCT04065399), with FLT3 inhibition as well as by performing knockdown of Menin using short hairpin RNAs, followed with Quizartinib treatment. The phosphorylation of FLT3 downstream signaling proteins such as STAT5 (pSTAT5) and ERK1/2 (pERK1/2) was also more affected by combined Menin-MLL and FLT3 inhibition compared to the single-drug treatment. Moreover, transcriptional profiling revealed a dramatic downregulation of STAT5A-dependent genes upon combinatorial treatment compared to single-drug or vehicle controls in MV411. Ectopic expression of Meis1 alone or together with Hoxa9 in the Npm1mut/+Flt3ITD/+ cells resulted in upregulation of Flt3 gene expression and partially rescued the anti-proliferative effect of both Menin-MLL inhibition alone and combined Menin-MLL and FLT3 inhibition. Primary AML cells harvested from patients with NPM1mut FLT3mut AML showed significantly better responses to combined Menin-MLL and FLT3 inhibition than to single-drug or vehicle control treatment while AML cells with wildtype NPM1, MLL, and FLT3 were not affected by any of the two drugs. The blast-colony formation of primary human leukemia cells, as well as murine Npm1mut/+Flt3ITD/+, was also most significantly abrogated with the drug combination versus all other treatment groups. Next, I evaluated the therapeutic effect of combined Menin-MLL and FLT3 inhibition in an MV411-derived leukemic xenograft murine model in vivo. I detected a significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment in mice treated with the combination of both inhibitors versus single-drug or vehicle treated animals. In a separate experiment, animals that had been treated with the combination of drugs also showed a significant survival advantage compared to the groups treated with a single drug or vehicle. In summary, combined Menin-MLL and FLT3 inhibition has synergistic on target activity against mutant FLT3 signaling in NPM1mut or MLL-r leukemias in vitro and in vivo. This concept may represent a novel therapeutic opportunity against these AMLs harboring a prognostically adverse FLT3-ITD, and is already available for clinical testing.
DDC-Sachgruppe:	000 Allgemeines 000 Generalities 500 Naturwissenschaften 500 Natural sciences and mathematics 570 Biowissenschaften 570 Life sciences 610 Medizin 610 Medical sciences
Veröffentlichende Institution:	Johannes Gutenberg-Universität Mainz
Organisationseinheit:	FB 10 Biologie
Veröffentlichungsort:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-5940
URN:	urn:nbn:de:hebis:77-openscience-f60cce06-89b4-4c00-b120-bd999b75a73b8
Version:	Original work
Publikationstyp:	Dissertation
Nutzungsrechte:	CC BY
Informationen zu den Nutzungsrechten:	https://creativecommons.org/licenses/by/4.0/
Umfang:	104 Blätter, Illustrationen, Diagramme
Enthalten in den Sammlungen:	JGU-Publikationen