MLL Rearrangements Research Articles

Dynamic assessment of WT1 for predicting the relapse of acute myeloid leukemia patients with MLL rearrangements after hematopoietic stem cell transplantation

Dynamic assessment of WT1 for predicting the relapse of acute myeloid leukemia patients with MLL rearrangements after hematopoietic stem cell transplantation

Small-Molecule Inhibition of the Acyl-Lysine Reader ENL as a Strategy against Acute Myeloid Leukemia.

AML is a poor-prognosis disease for which new therapeutic approaches are desperately needed. We developed an orally bioavailable inhibitor of ENL, demonstrated its potent efficacy in MLL-rearranged and NPM1-mutated AML, and determined its mechanisms of action. These biological and chemical insights will facilitate both basic research and clinical translation. This article is highlighted in the In This Issue feature, p. 2483.

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.

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease. Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease. Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%. Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases. Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

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.

MLL::AF9 degradation induces rapid changes in transcriptional elongation and subsequent loss of an active chromatin landscape

MLL rearrangements produce fusion oncoproteins that drive leukemia development, but the direct effects of MLL-fusion inactivation remain poorly defined. We designed models with degradable MLL::AF9 where treatment with small molecules induces rapid degradation. We leveraged the kinetics of this system to identify a core subset of MLL::AF9 target genes where MLL::AF9 degradation induces changes in transcriptional elongation within 15minutes. MLL::AF9 degradation subsequently causes loss of a transcriptionally active chromatin landscape. We used this insight to assess the effectiveness of small molecules that target members of the MLL::AF9 multiprotein complex, specifically DOT1L and MENIN. Combined DOT1L/MENIN inhibition resembles MLL::AF9 degradation, whereas single-agent treatment has more modest effects on MLL::AF9 occupancy and gene expression. Our data show that MLL::AF9 degradation leads to decreases in transcriptional elongation prior to changes in chromatin landscape at select loci and that combined inhibition of chromatin complexes releases the MLL::AF9 oncoprotein from chromatin globally.

Elucidating minimal residual disease of paediatric B-cell acute lymphoblastic leukaemia by single-cell analysis.

Minimal residual disease that persists after chemotherapy is the most valuable prognostic marker for haematological malignancies and solid cancers. Unfortunately, our understanding of the resistance elicited in minimal residual disease is limited due to the rarity and heterogeneity of the residual cells. Here we generated 161,986 single-cell transcriptomes to analyse the dynamic changes of B-cell acute lymphoblastic leukaemia (B-ALL) at diagnosis, residual and relapse by combining single-cell RNA sequencing and B-cell-receptor sequencing. In contrast to those at diagnosis, the leukaemic cells at relapse tended to shift to poorly differentiated states, whereas the changes in the residual cells were more complicated. Differential analyses highlighted the activation of the hypoxia pathway in residual cells, resistant clones and B-ALL with MLL rearrangement. Both in vitro and in vivo models demonstrated that inhibition of the hypoxia pathway sensitized leukaemic cells to chemotherapy. This single-cell analysis of minimal residual disease opens up an avenue for the identification of potent treatment opportunities for B-ALL.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

SUMMARYHOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of β-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced β-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

3077 – FUNCTIONAL AND MOLECULAR IDENTIFICATION OF A FETAL-ASSOCIATED TUMOR SUPPRESSOR AXIS: ABROGATION OF MLL-ENL DRIVEN ACUTE MYELOID LEUKEMIA

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML. MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML.

Effective Menin inhibitor-based combinations against AML with MLL rearrangement or NPM1 mutation (NPM1c)

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

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.

High Expression of BCL11A Predicts Poor Prognosis for Childhood MLL-r ALL.

BackgroundDespite much improvement in the treatment for acute lymphoblastic leukemia (ALL), childhood ALLs with MLL-rearrangement (MLL-r) still have inferior dismal prognosis. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy.MethodsGSE13159 and GSE28497 were selected via the Oncomine website. Differentially expressed genes (DEGs) between MLL-r ALLs and normal samples were identified by R software. Next, functional enrichment analysis of these DEGs were carried out by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Then, the key hub genes and modules were identified by Weighted Gene Co-expression Network Analysis (WGCNA). Therapeutically Applicable Research to Generate Effective Treatments (TARGET) ALL (Phase I) of UCSC Xena analysis, qPCR, and Kaplan-Meier analysis were conducted for validating the expression of key hub genes from bone marrow cells of childhood ALL patients or ALL cell lines.ResultsA total of 1,045 DEGs were identified from GSE13159 and GSE28497. Through GO, KEGG, GSEA, and STRING analysis, we demonstrated that MLL-r ALLs were upregulating “nucleosome assembly” and “B cell receptor signal pathway” genes or proteins. WGCNA analysis found 18 gene modules using hierarchical clustering between MLL-r ALLs and normal. The Venn diagram was used to filter the 98 hub genes found in the key module with the 1,045 DEGs. We identified 18 hub genes from this process, 9 of which were found to be correlated with MLL-r status, using the UCSC Xena analysis. By using qPCR, we validated these 9 hub key genes to be upregulated in the MLL-r ALLs (RS4;11 and SEM) compared to the non-MLL-r ALL (RCH-ACV) cell lines. Three of these genes, BCL11A, GLT8D1 and NCBP2, were shown to be increased in MLL-r ALL patient bone marrows compared to the non-MLL-r ALL patient. Finally, Kaplan–Meier analysis indicated that childhood ALL patients with high BCL11A expression had significantly poor overall survival.ConclusionThese findings suggest that upregulated BCL11A gene expression in childhood ALLs may lead to MLL-r ALL development and BCL11A represents a new potential therapeutic target for childhood MLL-r ALL.

Cytogenetic Characteristics of Childhood Acute Lymphoblastic Leukemia: A Study of 1541 Chinese Patients Newly Diagnosed between 2001 and 2014.

Cytogenetic abnormalities have been proven to be the most valuable parameter for risk stratification of childhood acute lymphoblastic leukemia (ALL). However, studies on the prevalence of cytogenetic abnormalities and their correlation to clinical features in Chinese pediatric patients are limited, especially large-scale studies. We collected the cytogenetics and clinical data of 1541 children newly diagnosed with ALL between 2001 and 2014 in four Chinese hospitals, and retrospectively analyzed their clinical features, prognosis and risk factors associated with pediatric ALL. All of these patients had karyotyping results, and some of them were tested for fusion genes by fluorescence in situ hybridization or reverse-transcription polymerase chain reaction. Overall, 930 cases (60.4%) had abnormal cytogenetics in this study, mainly including high hyperdiploidy (HHD, n=276, 17.9%), hypodiploidy (n=74, 4.8%), t(12;21)/TEL-AML1 (n=260, 16.9%), t(1;19)/E2A-PBX1 (n=72, 4.7%), t(9;22)/BCR-ABL (n=64, 4.2%), and t(v;11q23)/MLL rearrangements (n=40, 2.6%). The distribution of each cytogenetic abnormality was correlated with gender, age, white blood cell count at diagnosis, and immunophenotype. In addition, multivariate analysis suggested that t(v;11q23)/MLL rearrangements (OR: 2.317, 95%CI: 1.219-3.748, P=0.008) and t(9;22)/BCR-ABL (OR: 2.519, 95%CI: 1.59-3.992, P<0.001) were independent risk factors for a lower event-free survival (EFS) rate in children with ALL, while HHD (OR: 0.638, 95%CI: 0.455-0.894, P=0.009) and t(12;21)/TEL-AML1 (OR: 0.486, 95%CI: 0.333-0.707, P<0.001) were independent factors of a favorable EFS. The cytogenetic characteristics presented in our study resembled other research groups, emphasizing the important role of cytogenetic and molecular genetic classification in ALL, especially in B-ALL.

Preclinical Evaluation of a Novel Orally Bioavailable Menin-MLL Interaction Inhibitor, DSP-5336, for the Treatment of Acute Leukemia Patients with MLL-Rearrangement or NPM1 Mutation

Background: The 11q23 abnormalities involving mixed lineage leukemia (MLL) gene are frequently found in adult and pediatric patients with acute leukemia. MLL rearrangements (MLL-r) are often associated with a poor prognosis and show poor response to currently available therapies, thus developing more effective therapy is urgently required. The leukemogenic activity of MLL fusion proteins, the products of the chimeric genes of MLL and its fusion partners generated by MLL-r, is critically dependent on direct interaction with MENIN, the product of the MEN1 gene. Interaction of MLL fusion proteins with MENIN plays an important role to enhance the proliferation and to block the differentiation of hematopoietic cells by maintaining high expression of hematopoietic stem cell program genes, such as HOXA9 and MEIS1. It has also been reported that the MENIN interaction with wild-type MLL, is required to induce HOXA9 and MEIS1 expression and also crucial for the development of acute leukemia with nucleophosmin (NPM1) mutations. Therefore, the MENIN-MLL interaction inhibitor is expected to have anti-leukemogenic activity against acute leukemia with MLL-r or NPM1 mutation by suppressing the expression of HOXA9 and MEIS1 and inducing terminal differentiation.Results: We generated DSP-5336, a novel, potent, and orally bioavailable MENIN-MLL interaction inhibitor for the treatment of acute leukemia patients with MLL-r or NPM1 mutation. DSP-5336 directly bound to the MENIN protein (Kd = 6.0 nM) and inhibited the MENIN-MLL interaction (IC 50 = 1.4 ± 0.058 nM). DSP-5336 selectively inhibited the cell growth of human leukemia cell lines including MV-4-11, MOLM-13, KOPN-8, and OCI-AML3 (IC 50 = 10, 15, 31 and 15 nM, respectively). These DSP-5336-sensitive cell lines possess a MLL-r or NPM1 mutation. On the other hand, DSP-5336 did not affect the cell growth of human leukemia cell lines such as HL-60, MOLT-4, and Reh (IC 50 > 10 μM), which do not have MLL-r or NPM1 mutations. In a mouse xenograft model using MV-4-11 cells, which express MLL-AF4, DSP-5336 exhibited a significant antitumor activity at the doses of 25 mg/kg and 50 mg/kg, administered twice daily (BID) for 20 days. There were no dose related changes in general condition or body weight. The effects of DSP-5336 on the expression of MENIN-MLL-regulated genes and differentiation marker genes were evaluated using MV-4-11 cells as pharmacodynamics markers in vitro and in vivo. In both cases, DSP-5336 significantly reduced the gene expression of MEIS1 and HOXA9, representative leukemic genes regulated by the MENIN-MLL complex. On the contrary, DSP-5336 significantly increased the gene expression level of ITGAM, a terminal differentiation marker.The efficacy of DSP-5336 was further assessed in acute leukemia patient samples and in mouse AML models. DSP-5336 strongly inhibited blast colony formation and changed the gene expression of the pharmacodynamics markers (HOXA9, MEIS1 and ITGAM ) in an AML patient sample carrying the MLL-AF6 fusion. In patient-derived xenograft (PDX) model with NPM1 mutation, human CD45 positive cells in peripheral blood progressively decreased during and beyond the 28 day period of DSP-5336 administration at doses of 25, 50, and 100 mg/kg BID and achieved a complete remission with no relapse at the doses of 50 and 100 mg/kg BID. At these three dose levels, DSP-5336 also induced a significant prolongation of survival compared to the vehicle control. Similarly, in a PDX model with MLL-AF4, DSP-5336 induced complete remission and significant prolongation of survival at the doses of 100 mg/kg BID compared to the vehicle control. In mouse AML models wherein MLL-ENL- or MLL-AF10-transduced bone marrow cells are transplanted in syngeneic mice, DSP-5336 induced a significant prolongation of survival at the doses of 200 mg/kg once daily (QD) compared to the vehicle control and the standard chemo therapy (cytarabine+daunorubicin) group.Summary: DSP-5336 has a potential as an antitumor drug that provides survival advantages in acute leukemia patients with MLL rearrangement or NPM1 mutation. Currently, a Phase 1/2 clinical study of DSP-5336 is planned in AML and ALL patients. DisclosuresEguchi: Sumitomo Dainippon Pharma: Current Employment. Shimizu: Sumitomo Dainippon Pharma: Current Employment. Kato: Sumitomo Dainippon Pharma: Current Employment. Furuta: Sumitomo Dainippon Pharma: Current Employment. Kamioka: Sumitomo Dainippon Pharma: Current Employment. Ban: Sumitomo Dainippon Pharma: Current Employment. Ymamoto: Sumitomo Dainippon Pharma: Current Employment. Yokoyama: Sumitomo Dainippon Pharma: Research Funding. Kitabayashi: Sumitomo Dainippon Pharma: Research Funding.

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)

Phase 1/2, Open-Label, Dose Escalation, Dose Expansion Study of Menin Inhibitor DSP-5336 in Adult Patients (pts) with Acute Leukemia with and without Mixed-Lineage Leukemia (MLL)-Rearrangement or Nucleophosmin 1 (NPM1) Mutation

Relapsed or refractory acute leukemia (AL) in adults, including acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL), still represents a significant unmet need. Once AML or ALL relapses after initial treatment, the prognosis is extremely limited, with median survival <6 mo for AML and <1 y for ALL. This is especially true of AML or ALL with MLL rearrangements (MLLr) or relapsed AML with NPM1 mutations. Translocations of the MLL gene, found in approximately 5% to 10% of adult patients with AL, result in an aggressive subtype of leukemia, with a median overall survival (OS) of approximately 9 months and a very high rate of relapse after standard induction therapy for AML or ALL. The NPM1 mutation (NPM1m) is seen in approximately 30-35% of adult AML cases, most commonly in de novo AML, and is associated with a more favorable prognosis in newly diagnosed AML. However, this favorable prognostic effect is lost in the presence of concomitant FLT3-internal tandem duplication (ITD) mutations, which are seen in 40% to 50% of AML with mutated NPM1.These genomic alterations have been the focus for the development of DSP-5336, a novel small-molecule oral therapy that prevents binding of the menin protein to MLL fusion proteins that drive leukemogenesis.In nonclinical studies, DSP-5336 showed selective cell growth inhibitory activity against humanAL cell lines with MLLr or NPM1m. In addition, evidence of antitumor activity and survival advantage was demonstrated in 3 AML xenograft models with MLLr and/or NPM1m. Treatment with DSP-5336 may result in the reduction of leukemic cells, offering therapeutic benefits that include achieving complete remission and prolonged survival in patients with relapsed or refractory AML with MLLr or NPM1m or pts with relapsed or refractory ALL with MLLr.An open-label, single-arm, phase 1/2 study (NCT04988555) of DSP-5336 is being started to evaluate the safety and efficacy of DSP-5336 and determine the recommended phase 2 dose (RP2D) in adults aged ≥18 y with relapsed/refractory AML or ALL after at least one line of standard therapy, including hematopoietic stem cell transplant. Key inclusion criteria are ECOG performance status 0-2 and adequate cardiac, renal, and hepatic function. Patients on the strong CYP3A4 inhibitors ketoconazole, itraconazole, or isavuconazole, are excluded unless they can be safely taken off these medications or switched to another antifungal medication at least 7 days prior to start of study treatment.Patients will be dosed twice daily for 28 days per cycle. The study design is depicted below in Figure 1. In phase 1, 21-30 pts will be enrolled into multiple ascending dose cohorts (1-6 pts per dose level), with dose escalation determined using a two-parameter Bayesian logistic regression model. Determination of the RP2D will be informed by the maximum biologic effect or maximum tolerated dose, whichever is lower. The phase 2 study will include arm A (relapsed or refractory AML with MLLr) and arm B (relapsed or refractory AML with NPM1m) (Figure 1). Patients will be treated at the RP2D to evaluate clinical activity and safety, which will be monitored using Bayesian posterior probability to optimize enrollment with Bayesian stopping rules. Bayesian monitoring of responses will be started after the first 10 enrolled patients are evaluable for efficacy. Study endpoints are summarized in Table 1. [Display omitted] DisclosuresDaver: Genentech: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Hanmi: Research Funding; Trillium: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Novartis: Consultancy; ImmunoGen: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Glycomimetics: Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Novimmune: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Affinito: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Cai: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Dobrowolska: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Dow: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Eguchi: Sumitomo Dainippon Pharma: Current Employment. Song: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Stoudemire: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Watanbe: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment. Komarnitsky: Sumitomo Dainippon Pharma Oncology, Inc: Current Employment.

Essential Roles of Transcription Factor MEF2D in the Maintenance of MLL-Rearranged Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is an aggressive disease with uncontrolled proliferation of myeloid progenitor cells and a failure of proper cell differentiation. Chromosomal translocations of the KMT2A (MLL) gene are found in about 10% AMLs affecting both children and adults. Patients harboring MLL rearrangement (MLL-r) have a high relapse rate and poor overall survival, emphasizing an unmet need to understand MLL-r AML pathogenesis and develop new therapeutic means. Through a comparative analysis of super-enhancers in AML cell lines and primary samples, we discovered that MLL-r leukemia cells preferentially gain super-enhancer signals at the MEF2D gene. MEF2D is aberrantly activated in MLL-r AML patient samples and predicts poor disease outcomes, implying a potential oncogenic role of MEF2D. Indeed, depletion of MEF2D inhibits human MLL-r leukemia growth and induces profound differentiation. Mechanistically, MEF2D directly represses CEBPE, a master regulator of myeloid differentiation. CEBPE depletion could rescue the growth defect and cell differentiation induced by MEF2D loss. We also demonstrated that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced anti-leukemia effects. Interestingly, MEF2C, a target of MLL fusion oncoproteins, is by far the most studied MEF2 family member with essential roles in MLL-r AML. Our genetic, biochemical, and integrative genomic data shows that MEF2D and MEF2C, both upregulated in MLL-r AML, interact with each other, colocalize genome-widely, and co-regulate target gene expression. We are currently investigating the molecular mechanisms underlying the interdependent function between MEF2 paralogs in MLL-r AML. Collectively, our findings suggest that MEF2D is a novel transcriptional dependency in MLL-r AML and uncover the MEF2-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block. DisclosuresNo relevant conflicts of interest to declare.

Clinical-Stage Menin Inhibitor KO-539 Is Synergistically Active with Multiple Classes of Targeted Agents in KMT2A-r and NPM1-Mutant AML Models

The N-terminus of the histone-lysine-N-methyltransferase MLL1 (KMT2A) contains the menin binding domain (MBD). Menin is a scaffold protein that binds to MBD and tethers MLL1 to chromatin. The Menin-KMT2A complex regulates expression of the leukemogenic homeobox A9 (HOXA9) gene and its co-factor MEIS1 in myeloid stem progenitor cells. In MLL-rearranged (MLL-r) AML, the N-terminus of the KMT2A gene is fused to the C-terminus of any of over 80 fusion partners, most commonly AF4, AF9, ENL and ELL. These MLL fusion partners are components of (and recruit) the super elongation complex and DOT1L to induce H3K4Me3 and H3K79Me2 marks on active chromatin, driving aberrant expression of HOXA9, MEIS1, PBX3, MEF2C and CDK6. In AML with mutant NPM1 (NPM1c), the wild type menin-KMT2A complex is the main oncogenic driver of HOXA9, MEIS1 and FLT3, promoting self-renewal of mutant leukemic blasts. KO-539 is a novel, oral investigational drug candidate targeting the Menin-KMT2A protein-protein interaction. Preclinical data show that KO-539 and close analogs induce differentiation and loss of viability of AML cells, and exert profound anti-leukemic activity in multiple PDX models harboring MLL-FP or NPM1c when dosed continuously QD for 3-6 weeks. In an ongoing phase 1/2A KOMET-001 clinical trial (NCT04067336) evaluating KO-539 in adult patients with relapsed and/or refractory AML, significant biological activity as well as complete remission lacking minimal residual disease have been observed.The present studies were focused on further elucidating biologic effects and on determining synergistic activity of KO-539 with agents targeting BCL2, BET proteins and CDK6. Treatment with KO-539 dose-dependently inhibited in vitro growth, as well as induced differentiation (by both morphology and increased CD11b expression) and loss of viability of MOLM13 (MLL-AF9 and FLT3-ITD) and OCI-AML3 (NPM1c and homozygous NRAS mutation) cells and patient-derived (PD) MLL-r or NPM1c AML cells. This was associated with repression of MEIS1, PBX3, MEF2C, FLT3, MYC, BCL2 and CDK6 transcription but increased mRNA expression of ITGAM (CD11b). KO-539 treatment also dose-dependently depleted protein levels of Menin, MEIS1, FLT3, CDK6 and BCL2, but upregulated MCL1 and CD11b proteins in MOLM13 and OCI-AML3 cells. Mass cytometry (CyTOF) analysis of PD MLL-r and NPM1c AML samples confirmed that, following KO-539 treatment there was also decline in protein levels of Menin, MEIS1, MEF2C, PBX3, FLT3, CDK6 and BCL2 in phenotypically characterized AML stem cells (with high expression of CLEC12A, CD123, CD244, CD99, but low expression of CD11b). KO-539-mediated Menin depletion was associated with polyubiquitylation and proteasomal degradation of Menin protein, given that protein levels were restored by co-treatment with the proteasome inhibitor carfilzomib. Notably, co-treatment (in vitro) with KO-539 in combination with venetoclax, OTX015 (pan-BET inhibitor) or abemaciclib (CDK6 inhibitor) for 72 to 96 hours induced synergistic loss of viability in cultured cell lines and PD AML cells from both MLL-r and NPM1c AML but not normal CD34+ progenitor cells or AML cells lacking MLL-FP or NPM1c, as determined by the SynergyFinder algorithm. Findings of ongoing in vivo studies determining effects of treatment with KO-539 and/or venetoclax or OTX015, versus vehicle control in PDX models will be presented at the meeting. These preclinical findings highlight the molecular correlates of anti-AML efficacy of KO-539 and demonstrate potentially synergistic KO-539-based combinations with inhibitors of BCL2, BET proteins and CDK6 against MLL-r or NPM1 mutant AML. DisclosuresDaver: Bristol Myers Squibb: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Genentech: Consultancy, Research Funding; Glycomimetics: Research Funding; Amgen: Consultancy, Research Funding; Hanmi: Research Funding; Sevier: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Kadia: Aglos: Consultancy; AbbVie: Consultancy, Other: Grant/research support; Amgen: Other: Grant/research support; BMS: Other: Grant/research support; Cure: Speakers Bureau; Genentech: Consultancy, Other: Grant/research support; Dalichi Sankyo: Consultancy; AstraZeneca: Other; Astellas: Other; Genfleet: Other; Ascentage: Other; Cellonkos: Other; Sanofi-Aventis: Consultancy; Pulmotech: Other; Jazz: Consultancy; Liberum: Consultancy; Novartis: Consultancy; Pfizer: Consultancy, Other. DiNardo: Forma: Honoraria, Research Funding; Foghorn: Honoraria, Research Funding; Agios/Servier: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Novartis: Honoraria; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. Leoni: Kura Oncology: Current Employment. Burrows: Kura Oncology: Current Employment.

Clinical Outcomes of Patients with Newly Diagnosed Myelodysplastic Syndrome with MLL Aberrations

IntroductionMixed lineage leukemias (MLL) are an extremely aggressive subset of leukemias that are inherently resistant to several therapies. MLL aberrations (deletion KMT2A and MLL rearrangements) are extremely rare in myelodysplastic syndrome (MDS) and little is known regarding their clinical course or outcomes. The aim of the study is to determine the natural course of the disease in our patient population.MethodsA retrospective cytogenetic database search of patients presenting to a tertiary cancer center from January 2005 to February 2021 with the diagnosis of MDS and CMML with presence of MLL aberrations was conducted. Deletion KMT2A was defined as deletion of 11q23 on cytogenetic studies. Patients who had received prior therapy with hypomethylating agents (HMAs), cytarabine-based regimens, or intensive chemotherapy were excluded. The International Working Group 2006 criteria was used for response assessment. Progression-free survival (PFS) was calculated from date of diagnosis to date of progression from MDS to AML. Overall survival (OS) was determined from the date of diagnosis to the date of death or last follow-up visit.ResultsBetween January 2005 to February 2021, 3369 patients (pts) with the diagnosis of MDS (n=2761) and CMML (n=608) were identified. Of these, 30 pts had MLL aberrations with 18 pts with MLL rearrangements (MLL r) and 12 pts with deletion 11q23 (del-KMT2A). Baseline characteristics are present in Table 1.Among 18 pts with MLL r MDS, 6 pts did not undergo any therapy at our institution. Of the remaining 12, 9 (75%) pts received HMA based therapy, 1 (8%) patient (pt) proceeded to allogeneic stem cell transplant(allo-HSCT) and 2 (17%) pts received intensive chemotherapy (idarubicin, fludarabine and cytarabine). Of the 9 pts who received HMA therapy, 5 (56%) pts had transformation to AML, 2 (22%) pts achieved CR and underwent allo-HSCT, 1 (11%) pt had persistent disease and 1 (11%) developed concurrent metastatic sarcoma and stopped treatment. Out of the 2 (22%) pts who underwent allo-HSCT, 1 pt received maintenance HMA post-transplant but relapsed with transformation to AML after 9 months, and the other was lost to follow up. 1 (11%) pt who had persistent disease received intensive chemotherapy and went into CR and subsequently underwent double umbilical cord transplant and continues to remain in CR. The 2 (17%) pts who received intensive chemotherapy frontline both went into CR, but one developed concurrent malignancy and stopped treatment. The other pt was transitioned to HMA therapy but relapsed with transformation to AML. The 1 patient who received frontline allo-HSCT relapsed with transformation to AML after 5 months. The overall median follow-up duration was 24 months with a median OS of 10 months (Figure 1) and a median PFS of 3.5 months(Figure 2).12 pts with del-KMT2A were identified, out of which 2 pts did not receive treatment. Of the remaining 10 pts, 8 (80%) received HMA based therapy and 2 (20%) pts received investigational treatments. Of the 8 pts who received HMA therapy, 1 (12.5%) achieved CR and remains in CR, 4 (50%) pts had no response, 2 (25%) had hematological improvement (HI) but one died of other causes and the other elected to stop treatment, 1 (12.5%) pt had transformation to AML. Of the 4 pts with no response, 2 pts elected to stop treatment, with 1 developing concurrent malignancy. Of the remaining 2 pts, 1 pt was placed on an investigational agent and had progression to AML and the other was placed on lenalidomide and then an investigational agent, but ultimately pursued hospice. Of the 2 pts on investigational agents both had persistent disease and one patient died and the other elected to stop treatment due to concurrent illness. The overall median follow-up duration was 154 months with a median OS of 16 months (Figure 1) and median PFS of 8 months (Figure 2). Clinical course is presented in Table 2.ConclusionPrognosis of patients with MLL alterations and MDS is very poor. MLL r MDS had worse outcomes with rapid transformation to AML compared to del-KMT2A. New therapies are needed for this group of patients. [Display omitted] DisclosuresIssa: Syndax Pharmaceuticals: Research Funding; Novartis: Consultancy, Research Funding; Kura Oncology: Consultancy, Research Funding. Sasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Kantarjian: Novartis: Honoraria, Research Funding; Immunogen: Research Funding; Aptitude Health: Honoraria; Ascentage: Research Funding; Amgen: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; Jazz: Research Funding; Astellas Health: Honoraria; Astra Zeneca: Honoraria; Ipsen Pharmaceuticals: Honoraria; KAHR Medical Ltd: Honoraria; NOVA Research: Honoraria; Precision Biosciences: Honoraria; BMS: Research Funding; AbbVie: Honoraria, Research Funding; Taiho Pharmaceutical Canada: Honoraria.