MLL-rearranged Leukemia Research Articles

Inhibition of the Menin-MLL1 Interaction in MLL-Rearranged Primary Mixed-Phenotype Acute Leukemia Samples Promotes Leukemic Differentiation

Mixed phenotype acute leukemias (MPALs) simultaneously present features typical for more than one hematological cell lineage. The rarity of such patients combined with the lack of well-defined diagnostic criteria have so far prevented the design of optimal therapy regimens resulting in the occurrence of lineage switch and therewith associated treatment failures. Rearrangements involving MLL gene (also known as KMT2A) occur in around 15% of MPAL patients (typically infants) and are associated with a very poor outcome. Moreover, several studies demonstrated dependency of MLL-rearranged (MLL-r) leukaemias on the interaction between N-terminus of MLL with Menin and its successful therapeutic targeting by small molecular weight compounds, that are currently validated in clinical trials. Here we investigated potential application of the Menin-MLL inhibitor SNDX-5613 in MLL/AF4 MPAL infants who presented with the bilineage B/Myeloid phenotype. Using a co-culture approach with human bone marrow-derived mesenchymal stromal cells (MSCs) and media optimized for growth of lymphoid and myeloid lineages, we were able to expand and monitor primary MLL-r patient material for more than three weeks. Primary material from two MPAL patients and their lymphoid and myeloid subpopulations were analyzed for their drug responses including immunophenotypic and global transcriptomic characterization. We observed a dramatic loss of cell numbers in response to SNDX-5613, with the IC50 value ranging from 80 to 800nM. Similar analyses of the sorted fractions revealed that the observed loss of cell numbers is mainly caused by the reduction of the lymphoid subfraction. Immunophenotypic analyses showed that surviving cells within both lymphoid and myeloid fractions are losing CD34 with simultaneous gain of the CD38 marker, suggesting cellular maturation. Treatment of the lymphoid fraction with SNDX-5613 caused a dose-dependent loss of the CD19+CD33+ cell population and a simultaneous gain of CD19+CD33-, and CD19-CD33+ cells. These data suggest the cellular origin of the myeloid fraction to be residing within the lymphoid counterpart. Interestingly, myeloid cells responded to Menin-MLL inhibition with increased surface expression of monocytic markers (e.g. CD11b, CD14, CD15), elevated RNA expression of genes involved in cell adhesion and morphological changes, indicating myeloid differentiation of the surviving cells. Moreover, transcriptomic analysis of the lymphoid subfraction revealed dose-dependent increase of the MS4A1 gene expression (encoding for CD20) suggesting simultaneous lymphoid maturation of the CD19+ proB-like blasts. In conclusion, our study shows that Menin-MLL inhibition drives both myeloid differentiation and lymphoid maturation in MLL-r MPALs. These myeloid data are consistent with observations of differentiation syndrome as an on-target effect in current clinical trials while the phenotypic changes in the lymphoid cells are new findings. Together, these data strongly suggests that MLL-r MPAL patients could benefit from the inclusion of the Menin-MLL1 inhibitors into their treatment regimens.

The RNA Binding Protein Musashi-2 (MSI2) Sustains the Growth and the Leukemogenic Potential of MLL-Rearranged Acute Lymphoblastic Leukemia, and It Is Involved in Glucocorticoid Resistance

Acute Lymphoblastic Leukemia occurring in infants and carrying the rearrangement of KMT2A, aka MLL (MLLr infant ALL) is a rare but very aggressive leukemia typically associated with poor outcome. Patients suffer from a dismal prognosis (overall survival: 20-40%) mainly due to the resistance to conventional therapy and a high incidence of relapse. The biological mechanisms involved in the pathogenesis and disease progression of infant MLLr leukemia are not completely understood and the identification of druggable genes is urgently needed for the development of novel therapeutic strategies. The RNA-binding protein Musashi-2 (MSI2) is a post-transcriptional regulator of mRNA translation to proteins. MSI2 has a pivotal role in normal hematopoiesis and in a variety of tumors, including leukemia, where it is involved in cell proliferation, differentiation and maintenance of the (cancer) stem cell pool. Although several studies strikingly demonstrated the involvement of MSI2 in chronic and acute myeloid leukemia, with regards to B-ALL, few papers reported over-expression of MSI2 associated with a poor outcome in both pediatric and adult B-ALL patients; however functional data are still missing. By retrospectively analyzing the gene expression profile of a large cohort of Italian patients with pediatric leukemia (n=314) we observed that MSI2 is highly expressed across the different cytogenetic subgroups, and the level of expression is even higher in ALL compared to AML cases; thus reinforcing the hypothesis that MSI2 might have a crucial role also in ALL. To unravel the functional role of MSI2 in MLL-r ALL, we successfully generated a model of MSI2 knock-out (MSI2 KO) using CRISPR/CAS9 genome editing in the MLL::AF4+ ALL cell line SEM. In a long-term competitive assay in vitro, in which the MSI2 KO and CNTRL cells were co-cultured at the initial 90:10 ratio (KO:CNTRL), we observed that after 20 passages in culture the CNTRL counterpart overgrew the MSI2 KO cells (which indeed extinguished); thus demonstrating that the lack of MSI2 leads to a proliferation disadvantage. In agreement with this, MSI2 KO cells show an impaired leukemia-initiating capacity and a lower disease burden in vivo, and mice transplanted with MSI2 KO cells display a prolonged survival compared to controls. Also, we performed a high-throughput drug screening using a manually curated library of 174 compounds (FDA-approved, currently used in the clinic) and we observed that MSI2 KO cells show a marked sensitivity to Glucocorticoids (GCs). Further validation studies in vitro demonstrated that MSI2 KO cells were significantly (100-fold) more sensitive to Prednisolone or Dexamethasone than their MSI2-expressing counterpart. In addition, the treatment of SEM cells (typically resistant to GCs) with the MSI2 inhibitor Ro 08-2750 phenocopied the sensitization to GCs observed in CRISPR-edited MSI2 KO cells, showing synergistic effects in combination with Prednisolone or Dexamethasone. As infant MLLr ALL patients are typically resistant to GCs, our findings suggest that targeting MSI2 might represent a potential novel approach to circumvent the therapy resistance. Finally, by further investigating the biological mechanisms of GC-sensitization, we unraveled a potential role for MSI2 in the regulation of the energetic metabolism of leukemic cells. Indeed, the confocal analysis of mitochondrial membrane potential and the profiling of cellular bioenergetics by Seahorse technology revealed that MSI2 KO cells display mitochondrial hyperactivity at steady-state compared to controls. Upon GCs exposure (which inhibits glycolysis in both MSI2 KO and CNTRL cells) mitochondrial respiration is increased in CNTRL cells to compensate ATP production, while it is severely impaired in MSI2 KO, leading to a mitochondrial crisis and energy failure. The importance of metabolic plasticity of tumor cells in adaptation to stress conditions is emerging as a crucial aspect of cancer biology. The putative role of MSI2 in regulating the energy metabolism of leukemia is a novel finding not previously investigated, which may pave the way for targeting the metabolic vulnerabilities of ALL as a novel therapeutic approach. In conclusion, our study demonstrates that MSI2 sustains the growth in vitro and the leukemogenic potential in vivo of MLLr ALL cells, and it is involved in GC-resistance by regulating the energy metabolism of leukemia.

Genetic and Pharmacological Inhibition of TUT4/TUT7 Sensitizes AML Cells to BCL-2 Inhibition

The terminal uridyltransferases TUT4/7 (ZCCHC11/6) are important mediators of RNA stability, RNA decay, histone mRNA regulation, viral RNA sensing, and miRNA biogenesis. Regulation of RNA processing pathways can be mediated by a number of RNA-binding proteins (RBPs), and dysfunctional RNA processing has implications as a therapeutic target in leukemia and lymphoma. We performed genetic and pharmacological perturbation of the activity of terminal uridyltransferases TUT4/7 (ZCCHC11/6) in MLL-rearranged acute myeloid leukemia (AML). A combination of catalytic inactivation and knockout by CRISPR/Cas9 of TUT4 and TUT7, respectively, were used to abrogate uridylation in THP-1 cells, with enzyme function measured by a cellular miRNA uridylation assay. Inhibition of uridylation in THP-1 cells reduced proliferation, induced changes in cell cycle progression, and enhanced sensitivity to the BCL-2 inhibitor, venetoclax, alone and in combination with azacitidine. BCL-2 expression was found to be reduced at the mRNA and protein levels, while increases in expression of BCL-2 regulating miRNAs miR139-5p and miR494-3p were observed. GSEA performed on uridylation deficient cells highlighted changes in cell cycle regulation, mitochondrial metabolism, and cell death pathways relative to unedited controls. These effects could be recapitulated with pharmacological inhibition using sub-inhibitory doses of a novel, selective inhibitor of TUT4/7, TS-002266, while the weakly active enantiomer TS-002455 did not show combination activity. Treatment of primary AML with combination of TS-002266 and venetoclax improved activity over venetoclax alone in a number of primary AML samples. Thus, we propose that TUT4/7 and BCL-2 inactivation may be a therapeutic strategy in leukemia.

In This Issue

In This Issue

Discovery of Novel, Potent, and Selective Small-Molecule Menin-Mixed Lineage Leukemia Interaction Inhibitors through Attempting Introduction of Hydrophilic Groups.

Introduction of the N,N-dimethylaminoethoxy group to pyrido[3,2-d]pyrimidine led to the discovery of menin-mixed lineage leukemia (MLL) interaction inhibitor C20. C20 showed strong binding affinity to menin protein and achieved sub-micromolar potency in cell growth inhibition. C20 had good selectivity for the inhibition of the interaction between menin and MLL in the kinase profile evaluation. Pharmacokinetic studies demonstrated that C20 possessed good stability and low clearance rate in liver microsomes and acceptable bioavailability in rats. Subsequent oral administration of C20 showed potent antitumor activity in the MV4;11 subcutaneous xenograft models of MLL-rearranged leukemia. The docking study showed that C20 bound highly with menin, and the N,N-dimethylaminoethoxy group occupied the F9 pocket of menin. This study proved that introducing a hydrophilic group into the F9 pocket of menin would be a new strategy for the design of menin-MLL interaction inhibitors with potent binding affinity and improved physical properties.

Signal peptide-CUB-EGF-like repeat-containing protein 1-promoted FLT3 signaling is critical for the initiation and maintenance of MLL-rearranged acute leukemia.

A hallmark of mixed lineage leukemia gene-rearranged (MLL-r) acute myeloid leukemia that offers an opportunity for targeted therapy is addiction to protein tyrosine kinase signaling. One such signal is the receptor tyrosine kinase Fms-like receptor tyrosine kinase 3 (FLT3) upregulated by cooperation of the transcription factors homeobox A9 (HOXA9) and Meis homeobox 1 (MEIS1). Signal peptide-CUB-EGF-like repeat-containing protein (SCUBE) family proteins have previously been shown to act as a co-receptor for augmenting signaling activity of a receptor tyrosine kinase (e.g., vascular endothelial growth factor receptor). However, whether SCUBE1 is involved in the pathological activation of FLT3 during MLL-r leukemogenesis remains unknown. Here we first show that SCUBE1 is a direct target of HOXA9/MEIS1 that is highly expressed on the MLL-r cell surface and predicts poor prognosis in de novo acute myeloid leukemia. We further demonstrate, by using a conditional knockout mouse model, that Scube1 is required for both the initiation and maintenance of MLL-AF9-induced leukemogenesis in vivo. Further proteomic, molecular and biochemical analyses revealed that the membrane-tethered SCUBE1 binds to the FLT3 ligand and the extracellular ligand-binding domains of FLT3, thus facilitating activation of the signal axis FLT3-LYN (a non-receptor tyrosine kinase) to initiate leukemic growth and survival signals. Importantly, targeting surface SCUBE1 by an anti-SCUBE1 monomethyl auristatin E antibody-drug conjugate led to significantly decreased cell viability specifically in MLL-r leukemia. Our study indicates a novel function of SCUBE1 in leukemia and unravels the molecular mechanism of SCUBE1 in MLL-r acute myeloid leukemia. Thus, SCUBE1 is a potential therapeutic target for treating leukemia caused by MLL rearrangements.

Outcomes of allogeneic haematopoietic stem cell transplantation for paediatric patients with MLL-rearranged acute myeloid leukaemia

BackgroundThe presence of mixed-lineage leukaemia rearrangement (MLL-r) in paediatric patients with acute myeloid leukaemia (AML) is a poor prognostic predictor. Whether allogeneic haematopoietic stem cell transplantation (allo-HSCT) is beneficial in such cases remains unclear.MethodsWe evaluated the outcomes and prognostic factors of allo-HSCT in 44 paediatric patients with MLL-r AML in the first complete remission (CR1) between 2014 and 2019 at our institution.ResultsFor all the 44 patients, the 3-year overall survival (OS), event-free survival (EFS), and cumulative incidence of relapse (CIR) were 74.5%, 64.1%, and 29.1%, respectively. Among them, 37 (84.1%) patients received haploidentical (haplo)-HSCT, and the 3-year OS, EFS, and CIR were 73.0%, 65.6%, and 26.4%, respectively. The 100-day cumulative incidence of grade II–IV acute graft-versus-host disease (aGVHD) post-transplantation was 27.3%, and that of grade III–IV aGVHD was 15.9%. The overall 3-year cumulative incidence of chronic graft-versus-host disease (cGVHD) post-transplantation was 40.8%, and that of extensive cGVHD was 16.7%. Minimal residual disease (MRD)-positive (MRD +) status pre-HSCT was significantly associated with lower survival and higher risk of relapse. The 3-year OS, EFS, and CIR differed significantly between patients with MRD + pre-HSCT (n = 15; 48.5%, 34.3% and 59%) and those with MRD-pre-HSCT (n = 29; 89.7%, 81.4% and 11.7%). Pre-HSCT MRD + status was an independent risk factor in multivariate analysis.ConclusionsAllo-HSCT (especially haplo-HSCT) can be a viable strategy in these patients, and pre-HSCT MRD status significantly affected the outcomes.

Abstract 681: Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition

Abstract MLL1 (KMT2A) translocations lead to aberrant expression of stem cell associated gene programs in hematopoietic cells producing a particularly aggressive subtype of leukemia, namely MLL-rearranged acute myeloid and lymphoblastic leukemia (AML and ALL, respectively). In pediatric leukemia, the most common rearrangement is the t(9; 11) (p22; q23) reciprocal translocation, which results in the expression of the MLL-AF9 fusion gene. Since the MLL-AF9 rearrangement does not lead to direct activation of an enzyme, small molecule drug discovery remains challenging and despite decades of research on MLL-rearranged leukemia, we have been limited by the inability of acutely assessing the consequences of direct MLL-AF9 inactivation in relevant model systems. By coupling rapid PROTAC-mediated degradation of the oncogenic MLL-AF9 fusion protein with genome-wide gene expression and chromatin analyses we have now been able to identify the immediate transcriptional and chromatin state consequences of MLL-AF9 degradation. Specifically, we have established a core set of MLL-AF9 target genes whose expression changes within minutes of MLL-AF9 degradation. These rapid expression changes are mediated by changes in productive RNA Polymerase II elongation and increased RNA Polymerase II pausing. At later timepoints, degradation of the fusion protein also induces loss of an active chromatin landscape at MLL-AF9 target genes, characterized by loss of MLL-AF9 associated proteins and activating histone modifications. These insights improve our mechanistic understanding of how MLL-AF9 mediates chromatin remodeling and leukemogenesis, while also helping us evaluate small molecule inhibitors of epigenetic mechanisms that target the MLL-AF9 protein complex. Specifically, we investigated how degradation of MLL-AF9 compares to enzymatic inhibition of DOT1L or inhibition of the MENIN-MLL interaction. Interestingly, both DOT1L and MENIN inhibitors fell short when comparing their effects to MLL-AF9 degradation and only combined DOT1L/MENIN inhibition induced global MLL-AF9 destabilization on chromatin, thus disrupting the full MLL-AF9 directed epigenetic and transcriptional program. Ultimately, we were able to confirm these findings in a patient-derived xenograft model in vivo, where only the combination was able to significantly prolong leukemia free survival. In summary, our studies establish the cellular and molecular consequences of targeted degradation of the MLL-AF9 fusion oncoprotein and help improve our understanding of small molecule inhibitors of epigenetic mechanisms as cancer therapies. Citation Format: Sarah Naomi Olsen, Laura Godfrey, James P. Healy, Charles Hatton, Scott A. Armstrong. Targeted MLL-AF9 degradation is phenocopied by combined DOT1L and Menin inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 681.

Abstract 3943: Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML

Abstract Rearrangement of the mixed lineage leukemia (MLL) gene occurs in up to 10% of acute leukemias and is particularly prevalent in infant acute leukemias (~80% of infant ALL cases). MLL-rearranged (MLLr) leukemia is one of the high-risk types of leukemia with aggressive nature, resistance to therapy, and high frequency of early relapse, and currently there are no targeted therapies for its treatment. So, there is huge unmet medical needs for MLLr leukemias. The protein protein interaction (PPI) between menin and MLLr is critical to the pathogenesis of MLLr-driven leukemias by deregulation of the HOXA and MEIS1 genes. It has been demonstrated by both preclinical and clinical studies that blockage of this PPI with menin inhibitors has potential therapeutic effects on MLLr associated acute leukemias. NPM1 mutation (NMP1c) is a frequently mutated gene found in AML, and NMP1 mutated AML depends on menin-MLL interaction for leukemogenesis. Given the fact that wild type MLL and MLLr have similar leukemogenesis-driven target genes like HOXA and MEIS1, as well as the same binding pocket on menin, it was therefore conceived that menin inhibitor could be useful to the treatment of NPM-mutated AML, which was again supported by both preclinical and clinical data. Herein, we would like to report the discovery of our proprietary menin inhibitor. Taking advantage of a couple of key interactions with menin protein, our compounds has relatively low molecular weight for a challenging PPI target, however, demonstrated comparable to or even better potency than those menin inhibitors in clinical trials. Besides in vitro pharmacology data, the DMPK, PK/PD and in vivo efficacy in animal model will be discussed too. Citation Format: Taishan Hu, Zhilin Deng, Honghai Li, Xiaochu Ma, Quanrong Shen, Lei Zhang, Peihua Sun, Ye Hua, Byran Huang. Discovery of a novel menin-MLL inhibitor for potential treatment of MLLr leukemias and NPM1c AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3943.

Quantitative analysis of redox proteome reveals oxidation-sensitive protein thiols acting in fundamental processes of developmental hematopoiesis

Fetal and adult hematopoietic stem and progenitor cells (HSPCs) are characterized by distinct redox homeostasis that may influence their differential cellular behavior in normal and malignant hematopoiesis. In this work, we have applied a quantitative mass spectrometry-based redox proteomic approach to comprehensively describe reversible cysteine modifications in primary mouse fetal and adult HSPCs. We defined the redox state of 4,438 cysteines in fetal and adult HSPCs and demonstrated a higher susceptibility to oxidation of protein thiols in fetal HSPCs. Our data identified ontogenic changes to oxidation state of thiols in proteins with a pronounced role in metabolism and protein homeostasis. Additional redox proteomic analysis identified oxidation changes to thiols acting in mitochondrial respiration as well as protein homeostasis to be triggered during onset of MLL-ENL leukemogenesis in fetal HSPCs. Our data has demonstrated that redox signaling contributes to the regulation of fundamental processes of developmental hematopoiesis and has pinpointed potential targetable redox-sensitive proteins in in utero-initiated MLL-rearranged leukemia.

Discovery of a dual WDR5 and Ikaros PROTAC degrader as an anti-cancer therapeutic.

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40's non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.

JMJD1C-regulated lipid synthesis contributes to the maintenance of MLL-rearranged acute myeloid leukemia

Mixed Lineage Leukemia rearranged acute myeloid leukemia (MLLr AML) predicts a poor prognosis. Histone demethylase JMJD1C is a potential druggable target of MLLr AML. However, little is known about how JMJD1C contributes to MLLr AML. Here we found that JMJD1C regulates lipid synthesis-associated genes including FADS2, SCD in MLLr AML cells. Lipid synthesis-associated protein FABP5 was identified as a specific interacting protein of JMJD1C and binds to the jumonji domain of JMJD1C. FABP5 also regulates JMJD1C mRNA and protein expression. JDI-10, a small molecular inhibitor of JMJD1C identified by us, represses MLLr AML cells, induces apoptosis, and decreases JMJD1C-regulated lipid synthesis genes. Moreover, JDI-10 mediated suppression of MLLr AML cells can be rescued by palmitic acid, oleic acid, or recombinant FABP5. In summary, we identified that JMJD1C-regulated lipid synthesis contributes to the maintenance of MLLr AML. Lipid synthesis repression may represent a new direction for the treatment of MLLr AML.

Targeting matrix metallopeptidase 2 by hydroxyurea selectively kills acute myeloid mixed-lineage leukemia

Oncogene-induced tumorigenesis results in the variation of epigenetic modifications, and in addition to promoting cell immortalization, cancer cells undergo more intense cellular stress than normal cells and depend on other support genes for survival. Chromosomal translocations of mixed-lineage leukemia (MLL) induce aggressive leukemias with an inferior prognosis. Unfortunately, most MLL-rearranged (MLL-r) leukemias are resistant to conventional chemotherapies. Here, we showed that hydroxyurea (HU) could kill MLL-r acute myeloid leukemia (AML) cells through the necroptosis process. HU target these cells by matrix metallopeptidase 2 (MMP2) deficiency rather than subordinate ribonucleotide reductase regulatory subunit M2 (RRM2) inhibition, where MLL directly regulates MMP2 expression and is decreased in most MLL-r AMLs. Moreover, iron chelation of HU is also indispensable for inducing cell stress, and MMP2 is the support factor to protect cells from death. Our preliminary study indicates that MMP2 might play a role in the nonsense-mediated mRNA decay pathway that prevents activation of unfolding protein response under innocuous endoplasmic reticulum stress. Hence, these results reveal a possible strategy of HU application in MLL-r AML treatment and shed new light upon HU repurposing.

Quinazoline-Based Analog of Adenine As an Antidote Against MLL-rearranged Leukemia Cells: Synthesis, Inhibition Assays and Docking Studies

Background: Post-translational modifications of histones constitute a dynamic process impacting gene expression. A well-studied modification is lysine methylation. Among the lysine histone methyltransferases, DOT1L is implicated in various diseases, making it a very interesting target for drug discovery. DOT1L has two substrates, the SAM cofactor that gives the methyl group and the lysine H3K79 substrate. Results: Using molecular docking, the authors explored new bisubstrate analogs to enlarge the chemical landscape of DOT1L inhibitors. The authors showed that quinazoline can successfully replace the adenine in the design of bisubstrate inhibitors of DOT1L, showing similar activity compared with the adenine derivative but with diminished cytotoxicity. Conclusion: The docking model is validated together with the use of quinazoline in the design of bisubstrate inhibitors.

Potent, p53-independent induction of NOXA sensitizes MLL-rearranged B-cell acute lymphoblastic leukemia cells to venetoclax

The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells.

Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeler. Loss of SMARCB1 is associated with a complex set of phenotypic changes including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the ‘WIN’ site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the growth response of RT cells to WIN site blockade is independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to induce p53 and block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

3114 – FROM MECHANISMS OF LEUKAEMOGENIC DYSREGULATION TO NEW CANDIDATE THERAPEUTICS

Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities. Rearrangements involving the MLL1 gene (Kmt2a) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving MLL1 translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow HSPCs that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model implementing a conditionally blocked multipotent haematopoietic progenitor murine cell line (Hoxb8-FL) allows reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., et al 2019). Using our new cellular mouse model of AML, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Seq. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Thus, we have defined a leukaemic TF network consisting of around 60,000 links across approximately 11,000 genes. Previous work in our laboratory used the same approach to establish the gene regulatory function of 39 TFs in the wild-type parental Hoxb8-FL cells (Kucinski et al. 2020). Our model will allow us to directly compare our network to the wild-type cell of origin to interrogate how the TF network is underpinning leukaemic transcriptional dysregulation. Novel computational methods will allow us to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities.

CN470 is a BET/CBP/p300 multi-bromodomain inhibitor and has an anti-tumor activity against MLL-rearranged acute lymphoblastic leukemia

Acute lymphoblastic leukemia with chromosomal rearrangements involving the mixed-lineage leukemia (MLL) gene (MLL-r ALL) remains an incurable disease. Thus, development of a safe and effective therapeutic agent to treat this disease is crucial to address this unmet medical need. BRD4, a member of the bromodomain and extra-terminal domain (BET) protein family, and cyclic AMP response element binding protein binding protein (CBP) and p300, two paralogous histone acetyltransferases, are all considered cancer drug targets and simultaneous targeting of these proteins may have therapeutic advantages. Here, we demonstrate that a BET/CBP/p300 multi-bromodomain inhibitor, CN470, has anti-tumor activity against MLL-r ALL in vitro and in vivo. CN470, potently inhibited ligand binding to the bromodomains of BRD4, CBP, and p300 and suppressed the growth of MLL-r ALL cell lines and patient-derived cells with MLL rearrangements. CN470 suppressed mRNA and protein expression of MYC and induced apoptosis in MLL-r ALL cells, following a cell cycle arrest in the G1 phase. Moreover, CN470 reduced BRD4 binding to acetylated histone H3. The in vivo effects of CN470 were investigated using SEMLuc/GFP cells expressing luminescent markers in an orthotopic mouse model. Mice administered CN470 daily had prolonged survival compared to the vehicle group. Further, CN470 also showed anti-tumor effects against an MLL-r ALL patient-derived xenograft model. These findings suggest that inhibition of BET/CBP/p300 by the multi-bromodomain inhibitor, CN470, represents a promising therapeutic approach against MLL-r ALL.

The CDK4/6-UCHL5-BRD4 axis confers resistance to BET inhibitors in MLL-rearranged leukemia cells by suppressing BRD4 protein degradation

Among acute leukemias, mixed-lineage leukemia-rearranged (MLL-r) leukemia is associated with poor prognosis. Bromodomain and extra-terminal inhibitors (BETi) are promising agents for treatment of hematological malignancies; however, the mechanisms underlying sensitivity to BETi and biomarkers to predict sensitivity are yet to be clarified. Here, we established OTX015-resistant MLL-r cell lines (OTX015-R cells) and used them to explore therapeutic targets in BETi-resistant MLL-r leukemia. OTX015-R cells exhibited resistance to various BETi, and levels of bromodomain-containing protein 4 (BRD4) and BRD4-regulated molecules, such as c-MYC and B-cell/CLL lymphoma-2 (BCL-2), were remarkably increased in OTX015-R cells relative to those in the parental cells; however, BRD4 mRNA transcript levels were not elevated. These results suggest that overexpression of BRD4 protein, through suppression of BRD4 degradation, may contribute to BETi-resistance. Notably, expression of ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5) was increased in OTX015-R cells. Further, a UCHL5 inhibitor, b-AP15, and UCHL5 knockdown had antitumor effects by degrading BRD4. In addition, sensitivity to OTX015 was partially recovered in OTX015-R cells pretreated with b-AP15. Furthermore, cyclin-dependent kinase 4/6 (CDK4/6) inhibition decreased UCHL5 expression, suppressed OTX015-R cell proliferation, and induced apoptosis. These results indicate that the CDK4/6-UCHL5-BRD4 axis confers resistance to BETi by suppressing BRD4 degradation. We propose that this pathway is a potential novel therapeutic target in BETi-resistant MLL-r leukemia with BRD4 overexpression.

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)

Safety and Efficacy of Menin Inhibition in Patients (Pts) with MLL-Rearranged and NPM1 Mutant Acute Leukemia: A Phase (Ph) 1, First-in-Human Study of SNDX-5613 (AUGMENT 101)