Mixed Lineage Leukemia Fusion Research Articles

Blockade of the lncRNA-DOT1L-LAMP5 axis enhances autophagy and promotes degradation of MLL fusion proteins.

Mixed-lineage leukemia (MLL) fusion gene caused by chromosomal rearrangement is a dominant oncogenic driver in leukemia. Due to having diverse MLL rearrangements and complex characteristics, MLL leukemia treated by currently available strategies is frequently associated with a poor outcome. Therefore, there is an urgent need to identify novel therapeutic targets for hematological malignancies with MLL rearrangements. qRT-PCR, western blot, and spearman correction analysis were used to validate the regulation of LAMP5-AS1 on LAMP5 expression. In vitro and in vivo experiments were conducted to assess the functional relevance of LAMP5-AS1 in MLL leukemia cell survival. We utilized chromatin isolation by RNA purification (ChIRP) assay, RNA pull-down assay, chromatin immunoprecipitation (ChIP), RNA fluorescence in situ hybridization (FISH), and immunofluorescence to elucidate the relationship among LAMP5-AS1, DOT1L, and the LAMP5 locus. Autophagy regulation by LAMP5-AS1 was evaluated through LC3B puncta, autolysosome observation via transmission electron microscopy (TEM), and mRFP-GFP-LC3 puncta in autophagic flux. The study shows the crucial role of LAMP5-AS1 in promoting MLL leukemia cell survival. LAMP5-AS1 acts as a novel autophagic suppressor, safeguarding MLL fusion proteins from autophagic degradation. Knocking down LAMP5-AS1 significantly induced apoptosis in MLL leukemia cell lines and primary cells and extended the survival of mice in vivo. Mechanistically, LAMP5-AS1 recruits the H3K79 histone methyltransferase DOT1L to LAMP5 locus, directly activating LAMP5 expression. Importantly, blockade of LAMP5-AS1-LAMP5 axis can represses MLL fusion proteins by enhancing their degradation. The findings underscore the significance of LAMP5-AS1 in MLL leukemia progression through the regulation of the autophagy pathway. Additionally, this study unveils the novel lncRNA-DOT1L-LAMP5 axis as promising therapeutic targets for degrading MLL fusion proteins.

Design of Inhibitors That Target the Menin–Mixed-Lineage Leukemia Interaction

The prognosis of mixed-lineage leukemia (MLL) has remained a significant health concern, especially for infants. The minimal treatments available for this aggressive type of leukemia has been an ongoing problem. Chromosomal translocations of the KMT2A gene are known as MLL, which expresses MLL fusion proteins. A protein called menin is an important oncogenic cofactor for these MLL fusion proteins, thus providing a new avenue for treatments against this subset of acute leukemias. In this study, we report results using the structure-based drug design (SBDD) approach to discover potential novel MLL-mediated leukemia inhibitors from natural products against menin. The three-dimensional (3D) protein model was derived from Protein Databank (Protein ID: 4GQ4), and EasyModeller 4.0 and I-TASSER were used to fix missing residues during rebuilding. Out of the ten protein models generated (five from EasyModeller and I-TASSER each), one model was selected. The selected model demonstrated the most reasonable quality and had 75.5% of residues in the most favored regions, 18.3% of residues in additionally allowed regions, 3.3% of residues in generously allowed regions, and 2.9% of residues in disallowed regions. A ligand library containing 25,131 ligands from a Chinese database was virtually screened using AutoDock Vina, in addition to three known menin inhibitors. The top 10 compounds including ZINC000103526876, ZINC000095913861, ZINC000095912705, ZINC000085530497, ZINC000095912718, ZINC000070451048, ZINC000085530488, ZINC000095912706, ZINC000103580868, and ZINC000103584057 had binding energies of −11.0, −10.7, −10.6, −10.2, −10.2, −9.9, −9.9, −9.9, −9.9, and −9.9 kcal/mol, respectively. To confirm the stability of the menin–ligand complexes and the binding mechanisms, molecular dynamics simulations including molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) computations were performed. The amino acid residues that were found to be potentially crucial in ligand binding included Phe243, Met283, Cys246, Tyr281, Ala247, Ser160, Asn287, Asp185, Ser183, Tyr328, Asn249, His186, Leu182, Ile248, and Pro250. MI-2-2 and PubChem CIDs 71777742 and 36294 were shown to possess anti-menin properties; thus, this justifies a need to experimentally determine the activity of the identified compounds. The compounds identified herein were found to have good pharmacological profiles and had negligible toxicity. Additionally, these compounds were predicted as antileukemic, antineoplastic, chemopreventive, and apoptotic agents. The 10 natural compounds can be further explored as potential novel agents for the effective treatment of MLL-mediated leukemia.

Variable Response of MLL-Rearranged Leukemia Cell Lines to Combinations of Menin, CDK9 and DOT1L Inhibitors

Background: MLL (Mixed Lineage Leukemia) rearrangements occur in approximately 10% of leukemias. Infant leukemia has a higher frequency of MLL rearrangement (MLL-r) than leukemia in other age groups. Despite intensified treatment in infant leukemia, outcomes remain poor. MLL-r leukemia depends on the interaction of MLL fusions with menin, DOT1L, and CDK9. Single-agent treatment with menin, DOT1L, and CDK9 inhibitors has shown promising results in preclinical studies, but their clinical efficacy has been limited by primary and acquired resistance. We hypothesized that combination therapy targeting multiple regulators, including menin, DOT1L, and CDK9, would provide a more effective treatment of MLL-r leukemia. Methods: We performed cell viability experiments on leukemia cell lines (MV4;11, MOLM13, RS4;11, THP1, SEM, KOPN8, NOMO1, HL60, ML2) with single, two and three drug combinations using a menin inhibitor (VTP50469), a DOT1L inhibitor (EPZ5676), and a CDK9 inhibitor (AZD4573). The single drug treatment durations with VTP50469, EPZ5676, and AZD4573 were 10 days, 14 days, and 3 days, respectively. The number of viable cells was determined by trypan blue exclusion staining. After the combination treatment, cells were washed with PBS, placed in fresh media for recovery and counted on day 4 or 6. The effectiveness of the combination treatments was determined using SynergyFinder 3.0 web application. Annexin V/PI and CD11b staining were performed using flow cytometry. To demonstrate MLL-menin binding inhibition, we generated an N-terminal FLAG-tagged MLL expression construct in leukemia cell lines and performed co-immunoprecipitation and western blot analysis. Results:We first determined theIC50 values for each inhibitor separately. Based on the IC50 values of VTP50469 (menin inhibitor), we categorized MLL-r cell lines into three groups: very sensitive (MOLM13 and MV4;11), moderately sensitive (RS4;11 and SEM) and resistant (THP1 and ML2) [Fig. A]. Sensitivity followed a similar pattern (MOLM13 and MV4;11 -sensitive, THP1-resistant) when MLL-r cell lines were treated with EPZ5676 (DOT1L Inhibitor). In contrast, AZD4573 demonstrated consistent efficacy across all MLL-r leukemia cell lines, with THP1 and MOLM13 being particularly sensitive with IC50 values in the low nanomolar (nM) range. After identifying the IC50 values of each inhibitor, we performed viability experiments using two and three drug combinations. The combination of VTP50469 (menin inhibitor) and EPZ5676 (DOT1L inhibitor) was superior in decreasing cell viability in MLL-r leukemia cell lines sensitive to VTP50469 (MV4;11, MOLM13 and RS4;11) but not effective in THP1 cells which are resistant to VTP50469 and EPZ5676 [Fig. B]. Synergy scores analyzed using the ZIP model showed additive effects in MOLM13 and a synergistic response in MV4;11 and RS4;11. After 14-17 days of treatment with EPZ5676 combined with VTP50469, MV4;11, MOLM13 and RS4;11 cells exhibited no recovery from treatment, indicating a profound and persistent antileukemia effect of the combination treatment. Conversely, the combination of VTP5069 (menin inhibitor) and AZD4573 (CDK9 inhibitor) showed a synergistic inhibitory effect on cell viability only in THP1 [Fig. B]. Compared to the single-drug treatments, we observed an increased rate of apoptosis and differentiation in THP1 cells treated with the combination of VTP50469 and AZD4573. The three-drug combination of EPZ5676, VTP50469, and AZD4573 was not superior to the two-drug combinations in any of the MLL-r cell lines examined. To characterize VTP50469 resistance, we analyzed the inhibition of MLL-menin binding using an MLL expression construct in VTP50469 sensitive and resistant cell lines treated with VTP50469 doses ranging between 5-500 nM. Following treatment with VTP50469, the level of expression of the MLL construct correlated with the sensitivity of MLL-r leukemia cell lines to the menin inhibitor. Conclusions: Based on our preclinical in vitro findings, the co-inhibition of menin-DOT1L or menin-CDK9 is a promising approach for the treatment of MLL-r leukemia. The varying responses of different MLL-r leukemia cell lines to drug combination treatment indicate that not all cases of MLL-r leukemia will respond uniformly to treatment combinations. Taken together, our data provide a strategy to determine optimal treatment combinations for personalized treatment of MLL-r leukemia.

Synergistic Growth Inhibition of NPM1 Mutant AML PDX By Combined Therapy with BCL-2 Inhibitor Venetoclax (ABT-199) and Menin Inhibitor DS-1594b In Vivo

Introduction: Mixed-lineage-leukemia (MLL) translocations are prevalent in hematologic malignancies, resulting in aggressive leukemias resistant to standard therapies and low 5-year survival rates. These translocations lead to the production of MLL fusion proteins that promote leukemia development by enhancing proliferation, inhibiting hematopoietic differentiation, and driving leukemogenesis. Acute myeloid leukemia (AML), an aggressive blood cancer with a grim prognosis, particularly in elderly patients, frequently involves mutations in the nucleophosmin (NPM1) gene. Importantly, the interaction between Menin, MLL, and MLL fusion proteins play a crucial role in the pathogenesis of AML, both with MLL rearrangements (MLLr) and in leukemias carrying NPM1 mutations. Inhibition of the Menin-MLL interaction using novel Menin inhibitors has demonstrated efficacy in pre-clinical and clinical studies for both AML subsets. In this study, we investigate the effectiveness of the novel Menin-MLL inhibitor DS-1594b, developed by Daiichi Sankyo Co. Ltd., in combination with the BCL-2 antagonist venetoclax (ABT-199), in a patient-derived xenograft (PDX) model of NPM1-mutated acute leukemia. We have previously reported synergistic lethality and differentiation-inducing effects of this drug combination in both AML cell lines and primary leukemia samples in vitro (Ciaurro et al., Blood 2022). Our findings shed light on the potential of this therapeutic combination for the targeted treatment of AML with NPM1 mutations. Methods: NSG mice (female, 8-10 weeks old) were purchased from Jackson Laboratory. Mice were inoculated via the tail vein with NPM1m PDX/luc/GFP (3×10 6 cells). Once leukemia engraftment was confirmed by bioluminescent imaging mice were randomized in 4 groups: vehicle, ABT199, DS-1594b alone, and combination. After 12 days drugs were administered as follows: DS1594b at a dosage of 50 mg/kg by oral gavage for 4 weeks; ABT-199 at a dosage of 50 mg/kg by oral gavage for 2 weeks and 100mg/kg for the other 2 weeks. NSG mice were euthanized by CO 2 inhalation at the endpoint of the study in accordance with IACUC protocols. BM and spleens were harvested in a sterile environment and flushed in HBSS with 1% FBS. For CyTOF data spleen cells were initially barcoded and stained with metal-conjugated antibodies according to Fluidigm's protocols and samples were analyzed by CyTOF (Helios) Mass Cytometer. Results: New in vitro data confirmed synergistic lethality on additional AML cell lines and demonstrated upregulation of PARP-C and Caspase3-C apoptosis pathways using western blot analysis. Further, additional 3 primary AML patient samples showed additive or synergistic growth inhibition when treated with combination of drugs. In the in vivo experiment with NPM1m PDX/luc/GFP AML PDX model, we found that co-treatment with DS-1594b and venetoclax for four weeks resulted in a greater reduction in AML burden compared to treatment with either agent alone or a vehicle control as shown by Bioluminescence imaging. Moreover, weight of mice in the combo group was not significantly affected, while mice lost weight in vehicle and in single agent cohort due to rapid disease progression. The survival curve clearly demonstrated the benefits of the combination treatment, with the DS-1594b group having modest anti-tumor efficacy, and the combination group showing the greatest efficacy (Fig. 1A). To study the effects on cell differentiation, we used CyTOF technique, which allowed us to analyze a complete panel of 30 markers. We found two populations of cells, which we named populations (pop) 3 and pop 4, that were enriched in the group treated with the combination of drugs compared to the control group (Fig 1B). These populations of cells had higher levels of expression of CD33 and CD38, which are well-known markers of cellular differentiation. Conclusions: In this study, we present the in vivo effectiveness of DS-1594b in combination with venetoclax in a PDX model of NPM1-mutated AML. Moreover, the combination treatment exhibits differentiation-inducing effects, which contribute to its therapeutic effectiveness, and was safe. Overall, our findings strongly indicate that the combination of DS-1594b and venetoclax exhibits promising anti-leukemic activity in vivo and holds potential as a therapeutic strategy for AML patients with mtNPM1 mutations.

Discovery, Structure-Activity Relationship and In Vitro Anticancer Activity of Small-Molecule Inhibitors of the Protein-Protein Interactions between AF9/ENL and AF4 or DOT1L.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene cause 5-10% acute leukemias with poor clinical outcomes. Protein-protein interactions (PPI) between the most frequent MLL fusion partner proteins AF9/ENL and AF4 or histone methyltransferase DOT1L are drug targets for MLL-rearranged (MLL-r) leukemia. Several benzothiophene-carboxamide compounds were identified as novel inhibitors of these PPIs with IC50 values as low as 1.6 μM. Structure-activity relationship studies of 77 benzothiophene and related indole and benzofuran compounds show that a 4-piperidin-1-ylphenyl or 4-pyrrolidin-1-ylphenyl substituent is essential for the activity. The inhibitors suppressed expression of MLL target genes HoxA9, Meis1 and Myc, and selectively inhibited proliferation of MLL-r and other acute myeloid leukemia cells with EC50 values as low as 4.7 μM. These inhibitors are useful chemical probes for biological studies of AF9/ENL, as well as pharmacological leads for further drug development against MLL-r and other leukemias.

Unlocking the Mysteries of Chromatin Biology through Functional Epigenomics.

In the ever-evolving field of functional genomics, CRISPR-based screening technologies have become pivotal tools for elucidating gene function across various cell types. A recent study by Gilan and colleagues advances this technological frontier by introducing CRISPR-ChIP, a platform designed to investigate the complex dynamics of epigenetic regulation of chromatin. In proof-of-concept experiments, the authors demonstrate the potential of this tool to identify key molecular regulators of two major histone modifications associated with active transcription, H3 lysine 4 trimethylation (H3K4me3) and H3 lysine 79 dimethylation (H3K79me2). They further unveiled a previously unknown functional partitioning of the H3K79-specific methyltransferase DOT1L into an oncogenic complex with MLL-AF9 and a native complex with MLLT10, which cooperatively regulate Mixed Lineage Leukemia fusion protein (MLL-FP) target gene expression. This novel epigenomic approach integrates high-throughput CRISPR screening with ChIP-based direct readout of chromatin modifications in situ, offering a powerful tool to investigate the epigenetic regulatory layers across a diverse spectrum of biological processes and disease states.

Examination of in vitro and in vivo antitumor activity of Menin-MLL inhibitor, D0060-319.

e15119 Background: Rearrangements of the mixed-lineage leukemia (MLL) gene result in the production of MLL fusion proteins, which occurs in 5-10% of acute leukemia in adults and > 70% of infant leukemia, respectively. Menin protein, encoded by the MEN1 gene, interacts with MLL fusion proteins to induce leukemia. D0060-319, a Menin-MLL inhibitor, shows a significant antitumor effect in vivo and in vitro. Methods: The fluorescence polarization (FP) binding assay to assess inhibition of D0060-319 to Menin-MLL interaction. The cross reactivity of 44 molecular targets was tested to assess their off-target binding potential. The anti-proliferative ability of D0060-319 was evaluated by CCK-8 in different cell lines. The in vivo anti-tumor effect of D0060-319 was evaluated in a human MV4-11 and MOLM-13 cell mouse xenograft tumor model. Briefly, five million MV4-11 cells or 500 thousand MOLM-13 cells were subcutaneously implanted the right flank of CB-17 SCID mice to establish cell mouse xenograft tumor model. When tumor volume reached 100-120mm3, the animals were grouped and administrated with D0060-319 at the dose of 25, 50, 75, 100 mg/kg or vehicle at twice daily for 21days. Tumor measurements and body weights were performed twice weekly to assess the antitumor efficacy and tolerability of D0060-319. Results: D0060-319 competitively binds Menin to inhibit the interaction with MLL fusion protein with IC50 of 7.46nM, has high antiproliferative activity in MV4-11 and MOLM-13 cell lines containing MLL fusion proteins with IC50 values of 4.0nM and 1.7nM, respectively. However, in Kasumi-1, K562, HL-60 and KG-1 cell lines lacking MLL rearrangement, D0060-319 antiproliferative activity with IC50 > 10 μM, indicating selectivity for leukemia cell lines containing MLL fusion proteins active. In the MV4-11 cell mouse xenograft tumor model, D0060-319 significantly inhibited tumor growth at the dose of 25mg/kg, with a tumor growth inhibition (TGI) rate of 82.97%, and other doses (50, 75 and 100mg/kg) tumor completely regressed. In the MOLM-13 cell mouse xenograft tumor model, a dose-dependent anti-tumor effect was observed after 14 days of administration, with TGIs of 81.08%, 88.47% and 90.26% at the dose of 50, 75 or 100 mg/kg, respectively. In both CDX models, no bodyweight loss was observed, suggesting that D0060-319 was well tolerated. Furthermore, off-target screening assays showed that D0060-319 did not cross-react with any of the 44 molecular targets at 10 μM, indicating that D0060-319 is highly selective, high-affinity for the MLL-binding pocket on Menin. Conclusions: In MLL-rearranged leukemia, D0060-319 showed high antitumor activity and good tolerability both in vitro and in vivo.

Menin Inhibitor DS-1594b Drives Differentiation and Induces Synergistic Lethality in Combination with Venetoclax in AML Cells with MLL-Rearranged and NPM1 Mutation

Introduction: Mixed-lineage-leukemia (MLL) is one of the most frequently translocated genes in hematologic malignancies, which produces aggressive leukemias that are refractory to conventional treatment resulting in a 5-year survival rate of only 35%. MLL translocations lead to fusion of the N-terminal fragment of MLL with one of over sixty protein partners, producing MLL fusion proteins that enhance proliferation, block hematopoietic differentiation, and drive leukemia development. Acute myeloid Leukemia (AML) is a heterogeneous and rapidly progressing blood cancer with a poor prognosis, especially in elderly patients where it is more common. Mutations in the nucleophosmin (NPM1) gene are one of the most common causes of AML. Interestingly, the protein-protein interaction between Menin, MLL and MLL fusion proteins plays a critical role in the pathogenesis of acute myeloid leukemias, both with MLLr as well as in leukemias carrying mutations in NPM1. Inhibition of the MLL-Menin interaction with novel Menin inhibitors has shown efficacy in both AML subsets, in pre- clinical and clinical studies. However, the responses are short-lived and hence it is imperative to identify therapeutic approaches that may synergize with Menin inhibition. Our recent work showed that MLLr t(4;11) Acute Lymphoblastic leukemia () patient cells express high levels of the anti-apoptotic BCL-2 protein and are highly sensitive to treatment with the BCL-2 selective antagonist, venetoclax (Benito, J.M., et al , Cell Reports, 2015). Here, we report experiments to establish the efficacy of the novel Menin-MLL inhibitor DS-1594b which was developed by Daiichi Sankyo co., jp, in combination with the BCL-2 antagonist venetoclax (VEN) in MLLr and NPM1-mutated acute leukemias. Methods: The efficacy of DS-1594b/VEN combination in MLLr and NPM1mut AML cell lines and primary leukemia samples was evaluated by examining the cell viability in response to serial dilutions of tested compounds as single agents or in combination using Cell Titer Glo (CTG) assay at 3, 5 and 7 days of treatment. Induction of apoptosis was assessed by Annexin V/ staining. Since down-regulation of MLL fusion target genes leads to differentiation in MLL leukemic cells, we monitored cell differentiation by flow cytometry in AML cell lines (MLLr: OCI-AML2, MOLM13, MOLM14, MV411. NPM1 Mut: OCI-AML3 . Leukemia cell lines with no MLLr or NPM1 mutations: HL60, U937) and primary leukemia samples ( blood from relapsed MLLr and NPM1mut AML) using differentiation markers CD11b, CD14, CD15. MLLr cell line MOLM13 resistant to venetoclax was also included in these studies. Results: 7 days treatment with DS-1594b reduced viability in MLLr and NPM1mut cell lines at low nM concentrations but did not affect growth of negative control U937 and HL60 . DS-1594b induced differentiation in OCI-AML3 and Ven resistant cell after 5 days and in other lines after 10 days (figure 1A). DS-1594b did not induce apoptosis when used as a single agent in any AML cell line tested indicating that differentiation is a key mechanism of Menin inhibition. Five days of treatment with single agent showed a downregulation of Menin in all cell lines and downregulation of BCL2 in OCI-AML3 and OCI-AML2 in Western blot analysis. Remarkably, combination with Venetoclax induced synergistic lethality and growth inhibition in all cell lines. Most importantly, was observed in primary leukemia samples (Figure 1B), with differentiation effects after DS-1594b single agent (bottom). Conclusions: Here we show in vitro efficacy of DS-1594b, a selective small molecule menin inhibitor, as single agent or in combination with Venetoclax in MLLr and NPM1 mutated cell lines and primary leukemia samples. Preliminary data demonstrate differentiation effects of DS-1594b as single agent in both cell lines and primary AML leukemia samples. Combination effects show synthetic lethality in almost all cell lines except OCI-AML3. The combination elicited profound apoptosis in primary leukemia samples. In vivo studies using MLLr and NPM1 PDXs models are ongoing and will be presented. Overall, the preliminary findings presented here pave the way for further studies aimed at understanding mechanisms of and further development and testing of this combination in AML with MLLr and/or NMP1 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Design, Synthesis, and Biological Evaluations of DOT1L Peptide Mimetics Targeting the Protein-Protein Interactions between DOT1L and MLL-AF9/MLL-ENL.

On the basis of a previously identified DOT1L peptide mimetic (compound 3), a series of novel peptide mimetics were designed and synthesized. These compounds can potently bind to AF9 and ENL either in cell-free binding assays or in leukemia cells, and selectively inhibit the growth of leukemia cells containing mixed lineage leukemia (MLL) fusion proteins. The most potent compound 12 exhibited comparable anticancer cellular activities to those of EPZ5676, a clinical stage enzymatic inhibitor of DOT1L in several leukemia cell lines containing MLL fusion proteins. Mechanism studies for compound 12 indicated that it did not affect the global methylation of H3K79 catalyzed by DOT1L but could effectively suppress the methylation of H3K79 at MLL fusion proteins targeted genes and inhibit the expressions of these genes. Our studies thus demonstrated that inhibiting the protein-protein interactions between DOT1L and MLL fusion proteins is a potentially effective strategy for the treatment of MLL rearranged leukemias.

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.

Recent Progress of Small Molecule Menin-MLL Interaction Inhibitors as Therapeutic Agents for Acute Leukemia.

Mixed lineage leukemia (MLL) gene rearrangements are associated with acute leukemia. The protein menin is regarded as a critical oncogenic cofactor of the resulting MLL fusion proteins in acute leukemia. A direct interaction between menin and the MLL amino terminal sequences is necessary for MLL fusion protein-mediated leukemogenesis. Thus, inhibition of the interaction between menin and MLL has emerged as a novel therapeutic strategy. Recent improvements in structural biology and chemical reactivity have promoted the design and development of selective and potent menin-MLL interaction inhibitors. In this Perspective, different classes of menin-MLL interaction inhibitors are comprehensively summarized. Further research potential, challenges, and opportunities in the field are also discussed.

Altered Enhancer Activity Represses CEBPE Expression in MLL -Rearranged Leukemia

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins resulting from chromosomal translocation between MLL and its partner genes. The resultant fusion proteins can confer aberrant epigenetic patterns at gene regulatory elements to disrupt transcriptional program, and ultimately leads to differentiation blockage at early hematopoietic progenitor stage. CCAAT enhancer binding proteins (C/EBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. We found that Cebpe is markedly downregulated in murine hematopoietic stem/progenitor cells (HSPCs) expressing MLL-EEN, which could be associated with differentiation blockage in MLL -rearranged leukemia. However, how CEBPE is epigenetically dysregulated by MLL fusion proteins remains unclear. In murine granulocytes, we demonstrated long-range chromatin interaction between Cebpe promoter and enhancer by chromatin conformation capture (3C) assay, conferring the strong expression of Cebpe . Strikingly, we noticed gain of DNA methylation at Cebpe promoter in MLL-EEN expressing HSPCs, suggesting the disruption of chromatin interaction through aberrant epigenetic alterations resulted in Cebpe repression. Interestingly, CEBPE expression was also found significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). DNA hypermethylation was observed at both promoter and predicated enhancer regions in MLL AML cells, which were absent in non-MLL AML cells. In addition, reduction of H3K27ac enrichment at the predicted enhancer was found in MLL AML cells. Analysis of the public available RNA Polymerase II (RNAPII) ChIP-seq data demonstrated RNAPII binding at both CEBPE promoter and enhancers in non-MLL AML cell lines, but it was absent at the enhancer region in MLL AML cells. These results suggest a common epigenetic mechanism which disrupts the CEBPE enhancer-promoter interaction in MLL -rearranged leukemia. Besides, treatment of MLL AML cell lines with DNA methylation inhibitor 5'-aza-cytidine (5-aza-C) demonstrated induction of CEBPE and myeloid marker CD11b . While all -trans retinoid acid failed to trigger cell differentiation in ML-2 AML cells, co-treatment with 5-aza-C resulted in further induction of CEBPE and CD11b, compared to 5-aza-C treatment per se . Collectively, our work shows that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells. This study provides novel insights on the aberrant epigenetic regulation in MLL -rearranged leukemia and facilitates the development of differentiation therapy for leukemia. DisclosuresNo relevant conflicts of interest to declare.

The AF9-Binding Domain of DOT1L Is Critical for Its Recruitment By MLL Fusion Proteins in Leukemia and Contributes to Its Functions in Normal Hematopoiesis

Mixed lineage leukemia (MLL) is a very aggressive therapy resistant leukemia. MLL-AF9 is one of the most common MLL-fusion proteins that recruits disruptor of telomeric silencing 1-like (DOT1L), to MLL-target genes HOXA9/MEIS1. DOT1L is a histone methyltransferase that catalyzes methylation of H3K79, a gene activating mark, that is essential for leukemogenesis and normal hematopoiesis. Our lab and others mapped the 10 amino acid (865 - 874) binding site on DOT1L, which binds to the ANC1 homology domain (AHD) domain of AF9. We also identified a point mutation, I867A, sufficient to disrupt the AF9-DOT1L interaction in vitro. We demonstrated that DOT1L lacking the 10 amino acids (Δ10) were unable to support transformation by ML-AF9. We hypothesize that by disrupting the AF9-DOT1L protein-protein interaction (PPI), we will be able to inhibit leukemogenesis and potentially circumvent the negative consequences of DOT1L loss in normal bone marrow. We used a genetic approach to explore the role of DOT1L recruitment in leukemogenesis and normal hematopoiesis.We generated cell lines derived from MLL-AF9-driven DOT1Lf/f CreER(T2)+ bone marrow by transducing DOT1L constructs, including wild type (WT-DOT1L), deletion of 10 amino acids (Δ10-DOT1L), point mutant (I867A-DOT1L), and an enzymatically inactive mutant (RCR-DOT1L). Both Δ10 and I867A mutants inhibited leukemogenesis similarly to the RCR mutant. This inhibited proliferation was coupled with a decrease in H3K79me2, a decrease in HOXA9/Meis1 gene expression, cellular differentiation, and induction of apoptosis demonstrating that disruption of the AF9-DOT1L PPI mimics the phenotype of as the enzymatic inhibition, deeming it as a potential therapeutic target for this cancer.To explore the impact of disrupting the AF9-DOT1L interaction in normal bone marrow, we transduced Mx-Cre+ DOT1Lf/f bone marrow with either WT-DOT1L, Δ10-DOT1L, or RCR-DOT1L constructs and injected these cells into lethally irradiated B6-CD45.1 recipients. After hematopoietic reconstitution, recipients were treated with poly(I:C) to inactivate endogenous DOT1L. In the absence of DOT1L's methyltransferase activity (RCR-DOT1L), transduced hematopoietic progenitors underwent rapid failure. Interestingly, expression of the Δ10-DOT1L mutant was sufficient to support short-term hematopoiesis but insufficient to sustain long-term hematopoietic stem cells (LT-HSCs). Together these results show that blocking DOT1L's recruitment eliminates the transformative potential of the MLL-AF9 oncogene and that while DOT1L's function is critical for all hematopoietic progenitor cell survival, removing the DOT1L AF9 binding domain allows for short term hematopoiesis.To further explore the role of AF9 role in recruiting DOT1L in MLL-AF9 leukemia and normal hematopoiesis, using high throughput screening approach, we identified small molecules that bind to the AF9 AHD domain. These compounds inhibit proliferation of cells harboring the MLL-AF9 fusion, decrease H3K79me2, and induce apoptosis similar to Δ10-DOT1L and I867A-DOT1L mutants. We are currently working to further characterize their on-target activity in cells, and test their effects on normal bone marrow, with the ultimate goal to validate additional avenues for therapeutic intervention for MLL-rearranged leukemia. DisclosuresNo relevant conflicts of interest to declare.

Gain of Function MLL1 Model Systems Distinguish Hematopoietic from Leukemogenic Pathways

Hematopoiesis represents an ongoing developmental process in which hematopoietic stem (HSCs) or progenitor cells (HPCs) differentiate into various blood lineages in response to intra- and extra-cellular signals. Recent work has illuminated the centrality of epigenetic regulators in controlling both normal and malignant hematopoiesis. Among them, MLL1 (Mixed Lineage Leukemia-1), is a paradigmatic histone H3 lysine 4 methyltransferase that is essential for HSC maintenance and is closely associated with hematologic disorders and acute leukemia when disrupted by chromosomal translocations or rearrangements. Previous studies successfully employed loss-of-function alleles to demonstrate a critical role for MLL1 in HSC function, suggesting increased MLL1 may expand HSCs or HPCs. However, this hypothesis has never been tested due to the lack of a gain-of-function (transgenic) model. Furthermore, it has remained unclear how increased wild-type MLL1 might differ from leukemogenic MLL1 fusion proteins in influencing normal or aberrant hematopoiesis. Here we present mouse models in which increasing wild-type MLL1, but not MLL-ENL, in developing hematopoietic progenitors promotes hematopoietic colony frequency (CFU).To investigate the impact of increasing MLL1 levels in hematopoietic populations, we generated a Tet-inducible transgene by integrating a single copy of human wild-type MLL1 into the Col1a1 locus using the system developed by the Jaenisch group (Beard C. et al. 2006). Targeted embryonic stem cells (ESCs) were identified and induced expression of MLL1 was confirmed to be within physiologic levels. This mild induction of MLL1 protein did not increase histone H3, lysine 4 methylation levels, but did influence gene expression. The ESCs were then differentiated in vitro to form embryoid bodies (EBs) that recapitulate early development of all three germ layers. MLL1 induction generally did not affect EB proliferation or morphology. Representative gene expression patterns characteristic of each of the germ layers were not significantly changed by inducing MLL1 expression during all or defined periods of EB differentiation. Induction of MLL1 from day 4-6 did not affect the numbers of c-Kit+/CD41+ hematopoietic progenitors produced. However, MLL1-induced, c-Kit+/CD41+ hematopoietic progenitors exhibited increased myelo-erythroid colonies. Surprisingly, this increase did not occur through protection from apoptosis.Side-by-side comparison of MLL1 versus MLL-ENL induction (Ugale A. et al. 2014) illustrated that the promotion of hematopoietic colonies is a function of wild-type MLL1 but not the oncogenic MLL-ENL. MLL-ENL induction did not transform EB-derived hematopoietic progenitors to form leukemic colonies or acquire serial replating activity. Instead, progenitors in which MLL-ENL was induced ceased to replate. Comparison of hematopoietic progenitors cultured from MLL1 and MLL-ENL induced EBs showed distinct immunophenotypes, gene expression patterns and cellular processes affected.We report the first model in which the induction of wild-type MLL1 expression is achieved and demonstrate that enhancing MLL1 protein levels can selectively increase hematopoietic potential in developing EBs. MLL1 and MLL-ENL regulate distinct gene sets in hematopoietic progenitors that result in different outcomes in developing hematopoietic cells. These findings provide novel insight into how HSC regulators can be subverted by leukemogenic programs. In addition, the enhanced hematopoiesis resulting from increased wild-type MLL1 illustrates the potential for expanding HSCs for transplantation. DisclosuresNo relevant conflicts of interest to declare.

Leukemogenic Pathway of MLL-AF4 Fusion-Positive Acute Lymphoblastic Leukemia

Background Many hematologic malignancies, including leukemia, are caused by genetic abnormalities such as chromosomal translocation. These gene defects usually perturb differentiation and proliferation of hematopoietic stem/progenitor cells. As observed in most cancers, multiple gene defects are required to develop overt leukemia, and multi-step tumorigenesis is also the rule in leukemogenesis. Leukemia-specific fusion genes are usually generated early in leukemogenesis as essential genetic defects (1st hit) producing pre-leukemic stem cells. Additional genetic defect(s) occur in pre-leukemic stem cells, finally generating leukemic stem cells that expand to present overt leukemia. Infant acute lymphoblastic leukemia (ALL) are frequently characterized by chromosome translocations involving 11q23, resulting in the rearrangement of the mixed-lineage leukemia (MLL) gene and subsequent generation of MLL fusion genes. Amongst the > 50 genes that have been identified as fusion partners of MLL, fusion with AF4 is characteristically and commonly observed in infant ALL, representing a hallmark of poor prognosis. In most cases of ALL with MLL-AF4, the fusion gene originates in utero as a primary event. Although mutations in RAS and FLT3 are frequently observed in MLL-AF4 -positive ALL, recent genetic data showed significantly lower number of accompanying mutations, which suggested that 2nd hit mutations were not necessary for developing MLL fusion-positive leukemia. The necessity of 2nd hit mutations in leukemogenesis with MLL-AF4 is still under debate. Materials and Methods Several previous studies have modeled leukemic transformation by introducing fusion genes into hematopoietic stem/progenitor cells; however, reproduction of certain forms of ALL, such as MLL-AF4 -positive infant ALL, has been challenging. We used the immature hematopoietic cells derived from mouse embryonic stem (ES) cells as targets for MLL-AF4 -mediated transformation. The MLL-AF4 fusion gene was introduced into mouse ES cells to obtain ES cell clones with stable expression of MLL-AF4 . These ES cells were conditioned to generate embryoid bodies (EBs) and subsequently differentiated into hematopoietic lineages. Tie2-positive cells, which contain immature hematopoietic stem cells, were separated and transplanted into immunodeficient non-obese diabetic/Shi-scid, IL-2Rγ null (NOG) mice to investigate leukemia development. Retroviral insertional mutagenesis was used to introduce additional genetic defect(s) as candidates for tumor-promoting 2nd hit mutations. Purified Tie2-positive cells were transduced using the murine stem cell virus (MSCV) and transplanted into NOG mice. Results Mouse ES cells with stable expression of the MLL-AF4 fusion were viable and could proliferate and differentiate to produce EBs. However, EBs derived from MLL-AF4 -positive ES cells differentiated via the mesenchymal pathway rather than into the Tie2-positive hematopoietic lineage. Mice transplanted with Tie2-positive cells with MLL-AF4 expression did not develop leukemia within the observed period. However, addition of an additional genetic change by retroviral insertional mutagenesis accelerated tumorigenesis in the recipient mice. The MLL-AF4 -expressing Tie2-positive cells with insertional mutagenesis showed clonal expansion and formed mostly extra-medullary tumors with B220 expression, but did not fully develop into tumors of hematopoietic organs. Viral integration sites of these tumors were analyzed to determine additional genetic events inducing tumor formation. In the MLL-AF4 model, viral integration often caused overexpression of gene(s) located near the integration site. Conclusion Considering the in utero formation of MLL-AF4 harboring pre-leukemic stem cells in ALL, hematopoietic progenitor cells or immature fetal cells may be potential targets of leukemic transformation. Immature hematopoietic cells such as the Tie2-positive cells derived from ES cells could be suitable candidates for transformation with MLL-AF4 . Clonal expansion of MLL-AF4 -positive cells after introduction of retroviral insertional mutagenesis suggests the necessity of additional genetic change(s) that activate certain target gene(s) in leukemogenesis with MLL-AF4 . We are currently analyzing the roles of these genes in the leukemic transformation of MLL-AF4 -positive hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

A first-in-human phase I study of ATR inhibitor M1774 in patients with solid tumors.

TPS3153 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase plays a critical role in the DNA damage response by sensing and responding to DNA replication stress, and by inducing cell cycle arrest to prevent aberrant replication and mitotic catastrophe. Based on extensive preclinical and limited clinical evidence, ATR inhibition is a promising treatment strategy as monotherapy for patients with advanced tumors harboring synthetically lethal conditions, such as alternative lengthening of telomeres (ALT) and inactivating mutations in ARID1A and ATM. M1774 is a potent, selective, orally administered ATR inhibitor that has been shown to exert antitumor activity in patient-derived xenograft tumors and acute myeloid leukemia xenograft tumors that express the ATR inhibition sensitizing mixed lineage leukemia fusion protein. This study (NCT04170153) aims to evaluate the safety and tolerability, maximum tolerated dose, recommended dose for expansion (RDE) and pharmacokinetics (PK) of M1774 (part A1), the effect of food on M1774 PK (part A2), and the efficacy of M1774 in patients with tumors harboring selected mutations (part A3). An additional objective is to assess the pharmacodynamics of M1774 by measuring relative changes in baseline p-CHK1 and γ-H2AX expression in paired tumor biopsies and serial blood samples. Methods: Patients aged ≥18 years, with an Eastern Cooperative Oncology Group performance status ≤1, adequate baseline hematological, renal and hepatic function, and with locally advanced or metastatic disease refractory to standard therapy are eligible. Patients with tumors bearing loss-of-function (LoF) mutations (determined by site testing or a central trial assay) in ARID1A, ATM, or ATRX and/or DAXX as ALT status surrogate markers; and measurable disease according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, will be enrolled in part A3. In the dose escalation phase (part A1 [open]), 18–24 patients are due to receive M1774 at a starting dose of 5 mg once daily. Dose escalation is determined by the safety monitoring committee and guided by a Bayesian 2-parameter logistic regression model. The preliminary food assessment (part A2) will follow a randomized two-sequence two-period crossover design in which ≤12 patients will be randomized (1:1) to receive a single dose of M1774 on Day –7 at the RDE (determined in part A1) in either a fed or fasted condition. After the food assessment, patients will subsequently receive M1774 according to the part A1 dosing schedule. In the preliminary efficacy study (part A3), patients (n = 20–24 for each of the three planned cohorts) with tumors harboring LoF mutations in the genes for ARID1A, ATM, ATRX and/or DAXX, will receive M1774 at the RDE. The primary efficacy endpoint is overall response (RECIST). The study is open and currently recruiting. Patients have been enrolled to seven cohorts in part A1 with no DLTs observed; dose escalation is ongoing. Clinical trial information: NCT04170153.

Tet1 is not required for myeloid leukemogenesis by MLL-ENL in novel mouse models.

The Ten Eleven Translocation 1 (TET1) gene encodes an epigenetic modifying molecule that is involved in demethylation of 5-methylcytosine. In hematological malignancies, loss-of-function mutations of TET2, which is one of the TET family genes including TET1, are frequently found, while the mutations of TET1 are not. However, clinical studies have revealed that TET1 is highly expressed in some cases of the hematological malignancies including acute myeloid leukemia. Indeed, studies by mouse models using conventional Tet1 knockout mice demonstrated that Tet1 is involved in myeloid leukemogenesis by Mixed Lineage Leukemia (MLL) fusion gene or TET2 mutant. Meanwhile, the other study showed that Tet1 is highly expressed in hematopoietic stem cells (HSCs), and that deletion of Tet1 in HSCs enhances potential self-renewal capacity, which is potentially associated with myeloid leukemogenesis. To examine the role of Tet1 in myeloid leukemogenesis more precisely, we generated novel conditional Tet1-knockout mice, which were used to generate the compound mutant mice by crossing with the inducible MLL-ENL transgenic mice that we developed previously. The leukemic immortalization in vitro was not critically affected by conditional ablation of Tet1 in HSCs with the induced expression of MLL-ENL or in hematopoietic progenitor cells retrovirally transduced with MLL-ENL. In addition, the leukemic phenotypes caused by the induced expression of MLL-ENL in vivo was not also critically affected in the compound mutant mouse model by conditional ablation of Tet1, although we found that the expression of Evi1, which is one of critical target genes of MLL fusion gene, in tumor cells was remarkably low under Tet1-ablated condition. These results revealed that Tet1 was dispensable for the myeloid leukemogenesis by MLL-ENL, suggesting that the therapeutic application of Tet1 inhibition may need careful assessment.

Nucleoside and Non-Nucleoside DOT1L Inhibitors: Dawn of MLLrearranged Leukemia.

Herein, the underlying role of disruptor of telomeric silencing 1-like (DOT1L) as a therapeutic target for mixed-lineage leukemia (MLL)-rearranged is comprehensively clarified. DOT1L can be aberrantly recruited by an MLL fusion partner, thereby causing the over-expression, of several leukemia relevant genes and eventually leading to leukemia. As the unique histone methyltransferase (HMT), DOT1L possesses the function to specifically methylate H3K79, which was identified as a hallmark of active transcription. Accordingly, blockading of DOT1L has been recognized as an effective approach for cancer treatment. Currently, nucleoside DOT1L inhibitors have been developed successfully with the only EPZ5676 entering phase I clinical trial in 2013, which was validated as 'orphan drug' toward MLL-rearranged leukemia by FDA. In order to find compounds with better pharmacokinetic properties as DOT1L inhibitors, other types of non-nucleoside skeletons have also been reported successively.

The RNA Binding Protein IGF2BP3 Is Required for MLL-AF4 Mediated Leukemogenesis

Chromosomal rearrangements of the mixed-lineage leukemia (MLL) gene are observed in acute lymphoblastic leukemias (ALL), acute myeloid leukemias (AML), and in rare mixed-lineage leukemia. Despite recent progress in therapeutic approaches, patients with MLL-rearranged (MLLr) leukemias still have very poor outcomes and a high risk of relapse. Of more than 90 fusion partner genes, MLL-AF4 is the most common MLL fusion protein in patients. Previously, we found that the RNA binding protein IGF2BP3 was specifically overexpressed in MLL-rearranged B-ALL, and enforced expression in vivo led to a pathologic expansion of hematopoietic stem and progenitor cells resulting in B and myeloid cell leukocytosis in the periphery. However, the requirement of IGF2BP3 in MLL-AF4 mediated leukemogenesis remains to be determined. Utilizing our previously generated list of differentially regulated targets with IGF2BP3 knockdown and a published dataset of MLL-Af4 targets, we determined that transcripts modulated by IGF2BP3 showed significant enrichment for MLL-Af4-bound genes. Furthermore, we observed that MLL-AF4 directly binds to and transcriptionally induces IGF2BP3. We performed ChIP-PCR assays on RS4;11 and SEM cell lines, human B-ALL cell lines that carry the MLL-AF4 translocation, and determined that the region in the first intron of IGF2BP3 is strongly bound by MLL-AF4. Furthermore, we observed a dose-dependent increase in luciferase reporter activity when we co-transfected a dual-luciferase reporter vector containing the promoter region of IGF2BP3 with increasing levels of MLL-AF4 expressing retroviral vector. To determine the role of Igf2bp3 in MLL-Af4 driven leukemogenesis, we generated the first Igf2bp3 KO murine model. Surprisingly, Igf2bp3 KO mice maintain normal, steady-state hematopoiesis. However, in striking contrast, deletion of Igf2bp3 in the MLL-Af4 leukemia model, significantly increases the survival of MLL-Af4 transplanted mice and greatly attenuates the disease. Furthermore, Igf2bp3 deficiency significantly reduced the tumor burden and disease severity. We observed significant decreases in WBC counts, spleen weights, and infiltrating leukemic cells visualized in histopathological analysis of hematopoietic tissues and quantified by FACS analysis. Moreover, deletion of Igf2bp3 led to a leukemia-initiating cell (LIC) disadvantage in vivo, demonstrated by significantly reduced engraftment in primary transplanted mice and reconstitution of secondary serially transplanted mice. To identify the transcripts directly regulated by Igf2bp3 in the context of MLL-Af4 driven leukemia, we carried out enhanced crosslinking and immunoprecipitation (eCLIP) transcriptome analysis of MLL-Af4 transformed early stem and progenitor cells and primary cells purified from splenic tumors of MLL-Af4 leukemic mice. We discovered an IGF2BP3-regulated post-transcriptional operon governing leukemic cell survival and proliferation, in which mRNA targets include the Hoxa locus and numerous genes within the Ras signaling pathway. In our study, we provide evidence that Igf2bp3 is required for the initiation of MLL-Af4 driven leukemia. We determined that Igf2bp3 is necessary for the development of and function of MLL-Af4 LICs. Mechanistically, we show that Igf2bp3 binds to and modulates the expression of hundreds of critical target transcripts. In summary, we demonstrate that Igf2bp3 is a positive regulator of MLLr leukemogenesis by targeting Hoxa transcripts such as Hoxa9 and numerous Ras signaling pathway transcripts, thereby controlling multiple downstream effector pathways required for disease initiation and aggressiveness. Together, our findings identify IGF2BP3 as an important, potential therapeutic target in this disease. Disclosures No relevant conflicts of interest to declare.

The Development of Inhibitors Targeting the Mixed Lineage Leukemia 1 (MLL1)-WD Repeat Domain 5 Protein (WDR5) Protein- Protein Interaction.

Mixed Lineage Leukemia 1 (MLL1), an important member of Histone Methyltransferases (HMT) family, is capable of catalyzing mono-, di-, and trimethylation of Histone 3 lysine 4 (H3K4). The optimal catalytic activity of MLL1 requires the formation of a core complex consisting of MLL1, WDR5, RbBP5, and ASH2L. The Protein-Protein Interaction (PPI) between WDR5 and MLL1 plays an important role in abnormal gene expression during tumorigenesis, and disturbing this interaction may have a potential for the treatment of leukemia harboring MLL1 fusion proteins. In this review, we will summarize recent progress in the development of inhibitors targeting MLL1- WDR5 interaction.