The interaction of Menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and potential therapeutic opportunity against NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. Transcriptional profiling upon pharmacological inhibition of the Menin-MLL complex revealed specific changes in gene expression with downregulation of the MEIS1 transcription-factor and its transcriptional target gene FLT3 being most pronounced. Combining Menin-MLL-inhibition with specific small-molecule kinase inhibitors of FLT3-phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream to FLT3 signaling. The drug combination induced synergistic inhibition of proliferation as well as enhanced apoptosis and differentiation compared to single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary AML cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined Menin 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. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared to the single-drug and vehicle control groups. Our data suggest that combined Menin-MLL and FLT3-inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

Subtype selectivity represents ac hallenge in many drug discovery campaigns.Atypical example is the FK506 binding protein 51 (FKBP51), whichh as emerged as an attractive drug target. The most advanced FKBP51 ligands of the SAFit class are highly selective vs.F KBP52 but poorly discriminate against the homologs and off-targets FKBP12 and FKBP12.6. During am acrocyclization pilot study,w eo bserved that many of these macrocyclic analogs have unanticipated and unprecedented preference for FKBP51 over FKBP12 and FKBP12.6. Structural studies revealed that these macrocycles bind with anew binding mode featuring atransient conformation, which is disfavored for the small FKBPs.Using aconformation-sensitive assayweshow that this binding mode occurs in solution and is characteristic for this new class of compounds.T he discovered macrocycles are non-immunosuppressive,e ngage FKBP51 in cells,a nd blockt he cellular effect of FKBP51 on IKKa.O ur findings provide an ew chemical scaffold for improved FKBP51 ligands and the structural basis for enhanced selectivity.

The FK506‐binding protein 51 (FKBP51) is a high‐molecular‐weight immunophilin that emerged as an important drug target for stress‐related disorders, chronic pain, and obesity. It has been implicated in a plethora of molecular pathways but remains best characterized as a co‐chaperone of Hsp90 in the steroid hormone receptor (SHR) maturation cycle. However, the mechanistic and structural basis for the regulation of SHRs by FKBP51 and the usually antagonistic function compared with its closest homolog FKBP52 remains enigmatic. Here we review recent structural and biochemical studies of FKBPs as regulators in the Hsp90 machinery. These advances provide important insights into the roles of FKBP51 and FKBP52 in SHR regulation.

Subtyp-Selektivitäti st oft eine Herausforderung bei der Wirkstoffentwicklung. Ein Beispiel ist das FK506-bindende Protein 51 (FKBP51) als Wirkstoffziel. Die am weitesten fortgeschrittenen FKBP51-Liganden der SAFit-Klasse sind hochselektiv gegenüber FKBP52, differenzieren aber kaum gegenüber den eng verwandten Proteinen FKBP12 und FKBP12.6. Eine Makrozyklisierungsstudie ergab,d ass viele dieser makrozyklischen Analoga eine unerwartete,n euartige Präferenz fürF KBP51 gegenüber FKBP12 und FKBP12.6 haben. Strukturellen Studien zufolge weisen diese Makrozyklen einen neuen Bindungsmodus auf,m it einer transienten Protein-Konformation, die fürd ie kleinen FKBPs ungünstig ist. Mithilfe eines konformationssensitiven Assaysz eigen wir, dass dieser Bindungsmodus in Lçsung auftritt und charakteristisch fürdiese neue Verbindungsklasse ist. Die Makrozyklen sind nichti mmunsuppressiv,b inden FKBP51 in Zellen und blockieren die zelluläre Wirkung von FKBP51 auf IKKa.Die Ergebnisse liefern ein neues chemisches Gerüst fürverbesserte FKBP51-Liganden und die strukturelle Grundlage füre ine erhçhte Selektivität.

NPM1mutant (NPM1mut) and MLL1-rearranged (MLL-r) acute myeloid leukemias (AMLs) exhibit aberrant expression of HOX and MEIS1 transcription factors and commonly harbor an activating mutation in the receptor tyrosine kinase FLT3. Pharmacologic inhibition of the menin-MLL1 complex reverses leukemogenic gene expression including MEIS1 and FLT3 and represents a therapeutic opportunity for the treatment of these leukemias. Here, we investigate the contribution of the menin-MLL1 complex to leukemic FLT3 signaling and assess the therapeutic potential of dual menin-MLL1 and FLT3 targeting. First, we performed RNA sequencing to delineate transcriptional changes associated with menin-MLL1 inhibition (-i) using the small molecule inhibitor MI503 in NPM1mut and MLL-r AMLs (OCI-AML3 and MV411 cells). In both leukemias, we confirmed MEIS1 and its target gene FLT3 to be among the most significantly downregulated genes. These results were validated in several human cell lines of NPM1mut or MLL-r AMLs and in a genetically engineered murine AML model harboring an NPM1mut and an internal tandem duplication mutation in the FLT3 gene (Npm1mut/+Flt3ITD/+,now referred to as Npm1mutFlt3ITD). Allele-specific qPCR upon MI503 treatment confirmed profound suppression of the FLT3ITD allele present in the MLL-r MV411 and MOLM13 cells. Total FLT3 protein expression was also reduced upon MI503 treatment in all tested NPM1mut and MLL-r AMLs. Next, we assessed the therapeutic potential of combined menin-MLL1-i and FLT3-i in the FLT3-ITD positive MLL-r and NPM1mut leukemias. We found dramatic synergistic growth inhibition and substantially enhanced apoptosis when combining MI503 with the specific small molecule FLT3 inhibitor AC220 (Quizartinib) compared to monotreatment or vehicle control in the FLT3ITD positive MLL-r MV4-11 and MOLM-13 cells. Similar results were obtained when MI503 was combined with other specific FLT3 inhibitors - Crenolanib and Gilteritinib. As the murine Npm1mutFlt3ITD AML cells harbor the Flt3ITD F692L gatekeeper mutation that conveys drug resistance to most FLT3 inhibitors, we used Ponatinib as a combination partner for MI503 in these cells and found similar synergistic suppression of proliferation and colony formation. No drug sensitivity to single or combo treatment was observed in the human HL60 and NB4 cells, or the murine Hoxa9-Meis1 transformed cells (all wildtype for NPM1, MLL, FLT3). To investigate the mechanism of drug synergy we then performed immunoblotting of phosphorylated (activated) FLT3 (pFLT3). As expected, we observed reduced pFLT3 upon direct FLT3 inhibition with AC220 and found decreased total FLT3 and pFLT3 with MI503 monotreatment. Of note, we observed the most pronounced pFLT3 reduction with combo treatment, most likely reflecting the cooperative effect of direct pFLT3 inhibition with AC220 and transcriptional suppression of total FLT3 via MI503. Transcriptional profiling revealed most dramatic reduction of FLT3 downstream signature gene expression including MYC and MYC-dependent genes upon combinatorial treatment compared to single drug or vehicle controls. Ectopic expression of Meis1 or Hoxa9-Meis1 in the Npm1mutFlt3ITD cells led to increased Flt3 gene and protein expression and partially rescued the anti-proliferative effect of MI503 and combined menin-MLL1-i and FLT3-i. Next, we assessed the combo drug regimen in a cell line derived leukemic xenograft murine model in vivo. We found significantly reduced leukemia burden defined by bone marrow engraftment after 14 days of treatment within the combo versus the single drug or vehicle control animals. In a separate experiment, animals that had been treated with the drug combo in vivo had a dramatically enhanced survival compared to the control groups. We then evaluated single and combo drug effects on five primary human NPM1mutFLT3ITD AML patient samples in a human stroma cell co-culture model. The most profound anti-leukemic effect was again detected with the combo treatment compared to all other controls. In summary, we demonstrate synergistic on-target activity against mutant FLT3 signaling with combined menin-MLL1 and FLT3 inhibition in leukemias with NPM1mut or MLL-r in vitro and in vivo. This concept may represent a novel therapeutic opportunity against these AMLs harboring a prognostically adverse FLT3-ITD. Disclosures Vassiliou: Kymab Ltd: Consultancy, Other: Minor Stockholder; Oxstem Ltd: Consultancy; Celgene: Research Funding. Armstrong:Mana Therapeutics: Consultancy, Equity Ownership; Accent Therapeutics: Consultancy, Equity Ownership; OxStem Oncology: Consultancy, Equity Ownership; Syros Pharmaceuticals: Consultancy, Equity Ownership; C4 Therapeutics: Consultancy, Equity Ownership; Cyteir Therapeutics: Consultancy, Equity Ownership; Janssen: Research Funding; Novartis: Research Funding; AstraZeneca: Research Funding; Epizyme, Inc.: Consultancy, Equity Ownership; Imago Biosciences, Inc.: Consultancy, Equity Ownership. Kühn:Pfizer: Consultancy; ABBVIE: Consultancy; Daiichi Sankyo: Other: travel Support; Celgene: Other: travel support.

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Acute myeloid leukemias (AML) that are driven by MLL1 (KMT2A)-fusion proteins (MLL-f) or NPM1 mutations (NPM1mut) are both associated with aberrant expression of HOX and MEIS1 transcription factors and commonly harbor mutations in the gene encoding the receptor tyrosine kinase FLT3. Inhibition of the menin-MLL1 interaction has been shown to be a therapeutic opportunity in MLL-f driven leukemias and we recently demonstrated that this interaction is a dependency in NPM1mut AML. MI503 a specific small molecule menin-MLL1 inhibitor reduces dramatically cell growth and reverses leukemogenic gene expression including MEIS1 and FLT3. To determine global transcriptional changes associated with menin inhibition we performed RNA sequencing upon MI503 treatment in NPM1mut OCI-AML3 and MLL-f driven MV411 cells. MEIS1 and its putative target gene FLT3 were found to be among the most significantly downregulated genes. MEIS1 and FLT3 were consistently downregulated in various human and murine leukemia cell lines driven by MLL-f or NPM1mut. Allele specific qPCR confirmed profound downregulation of the mutant FLT3 allele in the MLL-f driven MV411 and MOLM13 cells as well as murine Npm1mut/+Flt3ITD/+ cells that all harbor a FLT3-ITD mutation. FLT3 surface expression was also substantially reduced upon MI503 treatment as assessed by FACS. Next, we assessed combinatorial menin- and FLT3 tyrosine kinase inhibition using MI503 and the 2nd generation FLT3 inhibitor AC220. The two drugs worked in a synergistic way to promote growth inhibition and enhanced apoptosis compared to single drug treatment or vehicle alone in MOLM13 and MV411 cells. HL60 and NB4 AML cells lacking NPM1mut, MLL-f, or FLT3-ITD showed no response. Drug synergism was also observed in the murine NPM1mut/+FLT3ITD/+ AML cells when combining MI503 with ponatinib a tyrosine kinase inhibitor with activity against the FLT3-ITD F692L resistance mutation that has been described in these cells. Of interest, ectopic expression of Hoxa9-Meis1 resulted in upregulation of Flt3 and rescued the antiproliferative effect of combined menin- and FLT3-inhibition. Combined menin- and FLT3 inhibition reduced FLT3 phosphorylation more than AC220 or MI503 alone most likely reflecting the joint effect of AC220 mediated inhibition of FLT3 phosphorylation and transcriptional FLT3 suppression via MI503. Transcriptional profiling revealed substantial silencing of FLT3 downstream signature genes including MYC upon combinatorial treatment. In vivo treatment of leukemic MV411 xenografts with combined MI503 and AC220 resulted in significantly enhanced reduction of leukemia burden that was observed with single drug treatment compared to vehicle controls. Altogether, our data show that simultaneous menin- and FLT3 inhibition has a synergistic effect against leukemogenic FLT3 signaling and may represent a novel therapeutic concept for MLL-f and NPM1mut driven AML with concomitant FLT3-ITD. Citation Format: Margarita M. Dzama, Martha C. Taubert, Kerstin Kunz, Johanna Rausch, Chun-Wei Chen, Annalisa Mupo, Matthias Theobald, Thomas Kindler, Richard P. Koche, George S. Vassiliou, Scott A. Armstrong, Michael W. Kühn. Therapeutic targeting of FLT3 mutations in AML via menin-MLL1 and FLT3 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3841.

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