MLL Target Genes Research Articles

Uncovering NOTCH1 as a Promising Target in the Treatment of MLL-Rearranged Leukemia.

MLL rearrangement (MLLr) is responsible for the development of acute leukemias with poor outcomes. Therefore, new therapeutic approaches are urgently needed. The NOTCH1 pathway plays a critical role in the pathogenesis of many cancers including acute leukemia. Using a CRISPR/Cas9 MLL-AF4/-AF9 translocation model, the newly developed NOTCH1 inhibitor CAD204520 with less toxic side effects allowed us to unravel the impact of NOTCH1 as a pathogenic driver and potential therapeutic target in MLLr leukemia. RNA sequencing (RNA-seq) and RT-qPCR of our MLLr model and MLLr cell lines showed the NOTCH1 pathway was overexpressed and activated. Strikingly, we confirmed this elevated expression level in leukemia patients. We also demonstrated that CAD204520 treatment of MLLr cells significantly reduces NOTCH1 and its target genes as well as NOTCH1 receptor expression. This was not observed with a comparable cytarabine treatment, indicating the specificity of the small molecule. Accordingly, treatment with CAD204520 resulted in dose-dependent reduced proliferation and viability, increased apoptosis, and the induction of cell cycle arrest via the downregulation of MLL and NOTCH1 target genes. In conclusion, our findings uncover the oncogenic relevance of the NOTCH1 pathway in MLLr leukemia. Its inhibition leads to specific anti-leukemic effects and paves the way for further evaluation in clinical settings.

Abstract IA016: Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention

Abstract MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. More than 90 different MLL translocation partners have been identified with the most common fusion partners, AF9, ENL, AF4, and AF10. AF9 and ENL are closely related members of the YEATS domain protein family and have been identified as part of several reported complexes including Super Elongation Complex (SEC) and DotCom Complex. AF9 and ENL, through their C-terminal hydrophobic domain, known as ANC1 Homology Domain (AHD), recruit the histone H3K79 methyltransferase hDOT1L to the MLL target genes, constitutively activating transcription of pro-leukemic targets. We have characterized the protein−protein interactions between hDOT1L and MLL fusion oncogenic proteins, AF9 and ENL, on the biochemical, biophysical, and functional level. This study mapped a 10 amino-acid region (aa 865− 874), highly conserved in hDOT1L from a variety of species, as an interacting site with the AHD domain of AF9/ENL proteins and demonstrated that these PPIs are essential for cellular immortalization mediated by MLL-AF9. Applying genetic approach and studying the effects of blocking the AF9-hDOT1L PPIs or hDOT1L enzymatic activity, it was demonstrated complete disruption of hDOT1L recruitment to critical target genes, Hoxa9 and Meis1, in cells expressing hDOT1L mutants with an impaired AF9 binding site. This was followed by significant decrease in H3K79me2 in the promoter regions of these genes, cell differentiation and induction of apoptosis. To identify the potential therapeutic benefits of this novel targeting approach, the effect of these different hDOT1L mutants was investigated on adult murine hematopoiesis. The in vivo studies demonstrated that genetic interventions that result in loss of hDOT1L enzymatic activity rapidly depletes hematopoietic stem and progenitor cells. In contrast, blocking the AF9-hDOT1L PPIs allowed for sustained hematopoiesis. These studies provided an important proof of concept for the potential therapeutic advantage of inhibiting the AF9-hDOT1L interaction and disrupting the integrity of the MLL-fusion complexes. In addition, these results demonstrated that hDOT1L recruitment can be abolished by a single point mutant (I867A), highlighting the crucial role of the hydrophobic interactions and giving insights for further drug discovery. We have reported the first peptidomimetics targeting PPIs between MLL-AF9 and hDOT1L, providing evidence for the druggability assessment of this novel potential therapeutic approach. The AF9/ENL C-terminal domain, is an intrinsically disordered protein, representing a challenge for structural studies. Applying X-ray crystallography, we have successfully determined the 3D structure of the AF9 AHD domain in complex with peptidomimetics establishing a platform for structure-based design of novel, optimized, potent inhibitors, the current focus of our research efforts. Citation Format: Zaneta Nikolovska-Coleska. Characterization of the protein-protein interactions involved in hDOT1L epigenetic regulation on biochemical, structural and functional level towards developing new therapeutic intervention. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr IA016.

Mutant NPM1 Is Recruited to MLL Target Genes Via Its Acidic Stretch and Nuclear Export Factor CRM1 and Regulates Oncogenic Transcription in Acute Myeloid Leukemia

Mutant NPM1 Is Recruited to MLL Target Genes Via Its Acidic Stretch and Nuclear Export Factor CRM1 and Regulates Oncogenic Transcription in Acute Myeloid Leukemia

Krüppel-like Factor 4 Supports the Expansion of Leukemia Stem Cells in MLL-AF9-driven Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy of the bone marrow with 5-year overall survival of less than 10% in patients over the age of 65. Limited progress has been made in the patient outcome because of the inability to selectively eradicate the leukemic stem cells (LSC) driving the refractory and relapsed disease. Herein, we investigated the role of the reprogramming factor KLF4 in AML because of its critical role in the self-renewal and stemness of embryonic and cancer stem cells. Using a conditional Cre-lox Klf4 deletion system and the MLL-AF9 retroviral mouse model, we demonstrated that loss-of-KLF4 does not significantly affect the induction of leukemia but markedly decreased the frequency of LSCs evaluated in limiting-dose transplantation studies. Loss of KLF4 in leukemic granulocyte-macrophage progenitors (L-GMP), a population enriched for AML LSCs, showed lessened clonogenicity and percentage in the G2/M phase of the cell cycle. RNAseq analysis of purified L-GMPs revealed decreased expression of stemness genes and MLL-target genes and upregulation of the RNA sensing helicase DDX58. However, silencing of DDX58 in KLF4 knockout leukemia indicated that DDX58 is not mediating this phenotype. CRISPR/Cas9 deletion of KLF4 in MOLM13 cell line and AML patient-derived xenograft cells showed impaired expansion in vitro and in vivo associated with a defective G2/M checkpoint. Collectively, our data suggest a mechanism in which KLF4 promotes leukemia progression by establishing a gene expression profile in AML LSCs supporting cell division and stemness.

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.

Elucidating the Importance of DOT1L Recruitment in MLL-AF9 Leukemia and Hematopoiesis.

Simple SummaryMLL-rearranged leukemia, driven by MLL-fusion proteins, is an aggressive, therapy-resistant leukemia found in >60% of infant leukemia and ~10% of adult leukemia. Studies have shown that inhibiting DOT1L enzymatic activity blocks leukemogenesis. However, DOT1L is critical for various normal cellular functions, including hematopoiesis. This study aimed to show that targeting the interaction between the MLL-AF9 fusion and DOT1L would inhibit leukemogenesis while sparing non-leukemic hematopoiesis. We found that disrupting the AF9-DOT1L interaction with a single point mutation was sufficient to impair leukemogenesis. We also demonstrate that genetic interventions that result in loss of DOT1L enzymatic activity in non-leukemic cells rapidly depletes hematopoietic stem and progenitor cells within 7–10 days; however, hematopoiesis was preserved when the AF9-DOT1L interaction was disrupted, leaving the enzymatic function intact. These studies are a proof of concept demonstrating the potential therapeutic advantage of inhibiting the AF9-DOT1L interaction and disrupting the integrity of the MLL-fusion complex.MLL1 (KMT2a) gene rearrangements underlie the pathogenesis of aggressive MLL-driven acute leukemia. AF9, one of the most common MLL-fusion partners, recruits the histone H3K79 methyltransferase DOT1L to MLL target genes, constitutively activating transcription of pro-leukemic targets. DOT1L has emerged as a therapeutic target in patients with MLL-driven leukemia. However, global DOT1L enzymatic inhibition may lead to off-target toxicities in non-leukemic cells that could decrease the therapeutic index of DOT1L inhibitors. To bypass this problem, we developed a novel approach targeting specific protein-protein interactions (PPIs) that mediate DOT1L recruitment to MLL target genes, and compared the effects of enzymatic and PPIs inhibition on leukemic and non-leukemic hematopoiesis. MLL-AF9 cell lines were engineered to carry mutant DOT1L constructs with a defective AF9 interaction site or lacking enzymatic activity. In cell lines expressing a DOT1L mutant with defective AF9 binding, we observed complete disruption of DOT1L recruitment to critical target genes and inhibition of leukemic cell growth. To evaluate the overall impact of DOT1L loss in non-leukemic hematopoiesis, we first assessed the impact of acute Dot1l inactivation in adult mouse bone marrow. We observed a rapid reduction in myeloid progenitor cell numbers within 7 days, followed by a loss of long-term hematopoietic stem cells. Furthermore, WT and PPI-deficient DOT1L mutants but not an enzymatically inactive DOT1L mutant were able to rescue sustained hematopoiesis. These data show that the AF9-DOT1L interaction is dispensable in non-leukemic hematopoiesis. Our findings support targeting of the MLL-AF9–DOT1L interaction as a promising therapeutic strategy that is selectively toxic to MLL-driven leukemic cells.

LEDGF/p75 is dispensable for hematopoiesis but essential for MLL-rearranged leukemogenesis

AbstractMixed lineage leukemia (MLL) represents a genetically distinct and aggressive subset of human acute leukemia carrying chromosomal translocations of the MLL gene. These translocations result in oncogenic fusions that mediate aberrant recruitment of the transcription machinery to MLL target genes. The N-terminus of MLL and MLL-fusions form a complex with lens epithelium-derived growth factor (LEDGF/p75; encoded by the PSIP1 gene) and MENIN. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both MENIN and LEDGF/p75 are required for efficient MLL-fusion–mediated transformation and for the expression of downstream MLL-regulated genes such as HOXA9 and MEIS1. In light of developing a therapeutic strategy targeting this complex, understanding the function of LEDGF/p75 in normal hematopoiesis is crucial. We generated a conditional Psip1 knockout mouse model in the hematopoietic compartment and examined the effects of LEDGF/p75 depletion in postnatal hematopoiesis and the initiation of MLL leukemogenesis. Psip1 knockout mice were viable but showed several defects in hematopoiesis, reduced colony-forming activity in vitro, decreased expression of Hox genes in the hematopoietic stem cells, and decreased MLL occupancy at MLL target genes. Finally, in vitro and in vivo experiments showed that LEDGF/p75 is dispensable for steady-state hematopoiesis but essential for the initiation of MLL-mediated leukemia. These data corroborate the MLL-LEDGF/p75 interaction as novel target for the treatment of MLL-rearranged leukemia.

The DOT1L inhibitor Pinometostat decreases the host-response against infections: Considerations about its use in human therapy

Patients with acute myeloid leukemia frequently present translocations of MLL gene. Rearrangements of MLL protein (MLL-r) in complexes that contain the histone methyltransferase DOT1L are common, which elicit abnormal methylation of lysine 79 of histone H3 at MLL target genes. Phase 1 clinical studies with pinometostat (EPZ-5676), an inhibitor of DOT1L activity, demonstrated the therapeutic potential for targeting DOT1L in MLL-r leukemia patients. We previously reported that down-regulation of DOT1L increases influenza and vesicular stomatitis virus replication and decreases the antiviral response. Here we show that DOT1L inhibition also reduces Sendai virus-induced innate response and its overexpression decreases influenza virus multiplication, reinforcing the notion of DOT1L controlling viral replication. Accordingly, genes involved in the host innate response against pathogens (RUBICON, TRIM25, BCL3) are deregulated in human lung epithelial cells treated with pinometostat. Concomitantly, deregulation of some of these genes together with that of the MicroRNA let-7B, may account for the beneficial effects of pinometostat treatment in patients with MLL-r involving DOT1L. These results support a possible increased vulnerability to infection in MLL-r leukemia patients undergoing pinometostat treatment. Close follow up of infection should be considered in pinometostat therapy to reduce some severe side effects during the treatment.

Combination of CYC065, a Second Generation CDK2/9 Inhibitor, with Venetoclax or Standard Chemotherapies - a Novel Therapeutic Approach for Acute Myeloid Leukaemia (AML)

Objectives: Over the last 50 years, there has been a steady improvement in the treatment outcome of AML. However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. CYC065, a novel CDK inhibitor, has pre-clinical efficacy in AML. The survival of AML blasts is dependent on MCL-1 which is depleted following treatment with CYC065. The aims of this study are: i) to demonstrate target inhibition and characterise the mechanism of action of CYC065 in vitro; ii) to assess the effects of CYC065 on growth and survival of AML cells; and iii) to assess a synergistic effect of CYC065 in combination with other targeted or chemotherapy agents on growth and survival of AML cell lines and primary human AML cells. Methods: Western blotting was performed to elucidate the mechanism of action. CYC065 effects on gene expression were studied in three AML cell lines: OCI-AML3 (NPM1 and DNMT3A mutations), MOLM-13 (MLL-AF9) and MV4-11 (FLT3-ITD, MLL-AF4) by qRT-PCR using Fluidigm® Biomark technology. The efficacy of CYC065 was explored in the three cell lines in parallel with washout studies. A synergistic effect of CYC065 in combination with venetoclax, cytarabine, or azacitidine was explored in the three AML cell lines, seven primary AML and three normal control samples using a variety of assays measuring cell viability, apoptosis and proliferation. Results: Inhibition of CDK9 by 1µM CYC065 treatment for 4h and 24h was demonstrated in AML cell lines by a reduction in phosphorylation of pSer2 RNA polymerase II, leading to inhibition of transcription and loss of transcripts with short half-lives. As expected from the proposed mechanism of action, gene expression was generally suppressed following CYC065 treatment. Early events (4h) included decreases in cell cycle regulators including CDK7 and CDK9, E2F1, CDC25C and PPP1R10, pro-survival molecules, including MCL-1, BCL-2 and XIAP, and the MLL target genes, MEIS1 and RUNX1. Decreases in MCL-1 mRNA were confirmed at the protein level by Western blotting and preceded the induction of apoptosis (PARP cleavage). In functional assays, clinically relevant CYC065 concentrations of 0.75µM, 0.5µM and 1µM, which induce 50% apoptosis, were selected for the OCI-AML3, MOLM-13 and MV4-11 cell lines, respectively, to combine with 0.0018-0.9µM of venetoclax, 0.016-0.47µM of cytarabine, or 2-5.3µM of azacitidine using the drug combination ratio obtained from synergy assays using CompuSyn software. Preliminary results showed a synergistic activity in all cell lines. Washout studies showed a slight recovery of cell viability at low concentrations but not at 1μM CYC065. High diversity of genetic mutations was observed in the seven primary AML samples (see Table). In general, moderate to marked synergistic effects were observed in apoptosis and active caspase-3 assays when CYC065 in combination with the three partners when clinically achievable concentrations were used. The more complex the molecular genetic lesions or complexity of karyotype, the less efficacious the combination therapy. In proliferation assays, CYC065/venetoclax showed a slight synergistic effect, whereas, CYC065/cytarabine and CYC065/azacitidine combinations showed a marked synergistic effect in which an increase in a percentage of cell division arrest was observed. Conclusions: CYC065 pulse treatment effectively induced apoptosis in AML cell lines in vitro. Target inhibition was confirmed by analysis of gene and protein expression and was accompanied by induction of apoptosis and cell cycle arrest. A synergistic effect of CYC065 in combination with venetoclax, cytarabine, or azacitidine was seen in AML cell lines and primary AML cells. These results highlight the potential of CYC065 in combination with venetoclax or standard chemotherapy agents for the treatment of AML. Table Disclosures Zheleva: Cyclacel Ltd: Employment, Equity Ownership, Patents & Royalties. Copland:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Cyclacel: Research Funding.

Functional diversity of inhibitors tackling the differentiation blockage of MLL-rearranged leukemia

IntroductionThe chromosomal rearrangements of the mixed-lineage leukemia gene MLL (KMT2A) have been extensively characterized as a potent oncogenic driver in leukemia. For its oncogenic function, most MLL-fusion proteins exploit the multienzyme super elongation complex leading to elevated expression of MLL target genes. High expression of MLL target genes overwrites the normal hematopoietic differentiation program, resulting in undifferentiated blasts characterized by the capacity to self-renew. Although extensive resources devoted to increased understanding of therapeutic targets to overcome de-differentiation in ALL/AML, the inter-dependencies of targets are still not well described. The majority of inhibitors potentially interfering with MLL-fusion protein driven transformation have been characterized in individual studies, which so far hindered their direct cross-comparison.MethodsIn our study, we characterized head-to-head clinical stage inhibitors for BET, DHODH, DOT1L as well as two novel inhibitors for CDK9 and the Menin-MLL interaction with a focus on differentiation induction. We profiled those inhibitors for global gene expression effects in a large cell line panel and examined cellular responses such as inhibition of proliferation, apoptosis induction, cell cycle arrest, surface marker expression, morphological phenotype changes, and phagocytosis as functional differentiation readout. We also verified the combination potential of those inhibitors on proliferation and differentiation level.ResultsOur analysis revealed significant differences in differentiation induction and in modulating MLL-fusion target gene expression. We observed Menin-MLL and DOT1L inhibitors act very specifically on MLL-fused leukemia cell lines, whereas inhibitors of BET, DHODH and P-TEFb have strong effects beyond MLL-fusions. Significant differentiation effects were detected for Menin-MLL, DOT1L, and DHODH inhibitors, whereas BET and CDK9 inhibitors primarily induced apoptosis in AML/ALL cancer models. For the first time, we explored combination potential of the abovementioned inhibitors with regards to overcoming the differentiation blockage.ConclusionOur findings show substantial diversity in the molecular activities of those inhibitors and provide valuable insights into the further developmental potential as single agents or in combinations in MLL-fused leukemia.

PF219 THE LEDGF/P75 HOMOLOG HRP2 IS NOT IMPORTANT FOR THE INITIATION OF MLL‐REARRANGED LEUKEMIA

Background:HDGF‐related protein 2 or HRP2 is a member of the Hepatoma‐Derived Growth Factor‐related protein family. Through its N‐terminal PWWP domain it associates with methylated histone tails and functions as such as a chromatin reader. HRP2 also features an integrase binding domain (IBD), known for its interaction with cellular and viral proteins. Little is known about the cellular function of HRP2, yet it has been linked to the malignant hepatocellular carcinoma. HRP2 has mainly been studied in comparison with its paralog, Lens Epithelium Derived Growth Factor (LEDGF/p75), suggesting similar but not identical physiological roles for both proteins. Previous publications showed an important role for LEDGF/p75 in the development of MLL‐rearranged leukemia (MLL‐r). Interaction between the IBD domain of LEDGF/p75 and the N‐terminal part of an oncogenic MLL fusion protein results in aberrant expression of MLL target genes, which in turn are important for normal hematopoiesis.Aims:Since HRP2 is functionally related to LEDGF/p75 but its potential role in MLL‐r remains unknown, we aimed to investigate a potential role for HRP2 in MLL‐r leukemia.Methods:Protein‐protein interactions were investigated with immunoprecipitation and detected by western blot. Interactions were confirmed using recombinant proteins in Alpha Screen and NMR experiments. The role of HRP2 in normal hematopoiesis was investigated using a systemic HRP2 knockout mouse model. The role of HRP2 in mixed lineage leukemia was determined using lentiviral‐mediated knock down and rescue experiments in, and colony forming assay with human cell lines. In addition, we performed colony forming assay and bone marrow transplantation with primary mouse bone marrow cells, transduced with an MLL‐fusion containing vector.Results:We show for the first time that HRP2 directly interacts with MLL with similar affinities compared to LEDGF/p75. In addition, NMR analysis indicates a similar interface between MLL and either IBD domain. However, the HRP2‐MLL interaction occurs independently of MENIN, the stabilizing adaptor of the MLL‐LEDGF/p75 interaction. The systemic HRP2 knockout mice survived with wild type blood counts. Only upon serial plating of hematopoietic stem cells, a decrease in proliferation‐potential of HRP2 knockout cells was observed. Knocking down HRP2 in leukemic cell lines and primary murine MLL‐transformed cells resulted in a decreased transformation‐potential. In contrast, Hrp2 knockout cells from the systemic knock out mice were efficiently transformed by MLL fusions, suggesting that Hrp2 might be dispensable for the initiation of MLL‐rearranged leukemia.Summary/Conclusion:In summary, we report for the first time that HRP2 is able to interact with MLL, yet in a MENIN‐independent manner. However, our results suggest that HRP2 might not be important for the initiation of mixed lineage leukemia, pointing towards a different role for HRP2 and its homolog LEDGF/p75 in MLL‐r.

RNA Polymerase II-Dependent Transcription Initiated by Selectivity Factor 1: A Central Mechanism Used by MLL Fusion Proteins in Leukemic Transformation.

Cancer cells transcribe RNAs in a characteristic manner in order to maintain their oncogenic potentials. In eukaryotes, RNA is polymerized by three distinct RNA polymerases, RNA polymerase I, II, and III (RNAP1, RNAP2, and RNAP3, respectively). The transcriptional machinery that initiates each transcription reaction has been purified and characterized. Selectivity factor 1 (SL1) is the complex responsible for RNAP1 pre-initiation complex formation. However, whether it plays any role in RNAP2-dependent transcription remains unclear. Our group previously found that SL1 specifically associates with AF4 family proteins. AF4 family proteins form the AEP complex with ENL family proteins and the P-TEFb elongation factor. Similar complexes have been independently characterized by several different laboratories and are often referred to as super elongation complex. The involvement of AEP in RNAP2-dependent transcription indicates that SL1 must play an important role in RNAP2-dependent transcription. To date, this role of SL1 has not been appreciated. In leukemia, AF4 and ENL family genes are frequently rearranged to form chimeric fusion genes with MLL. The resultant MLL fusion genes produce chimeric MLL fusion proteins comprising MLL and AEP components. The MLL portion functions as a targeting module, which specifically binds chromatin containing di-/tri-methylated histone H3 lysine 36 and non-methylated CpGs. This type of chromatin is enriched at the promoters of transcriptionally active genes which allows MLL fusion proteins to selectively bind to transcriptionally-active/CpG-rich gene promoters. The fusion partner portion, which recruits other AEP components and SL1, is responsible for activation of RNAP2-dependent transcription. Consequently, MLL fusion proteins constitutively activate the transcription of previously-transcribed MLL target genes. Structure/function analysis has shown that the ability of MLL fusion proteins to transform hematopoietic progenitors depends on the recruitment of AEP and SL1. Thus, the AEP/SL1-mediated gene activation pathway appears to be the central mechanism of MLL fusion-mediated transcriptional activation. However, the molecular mechanism by which SL1 activates RNAP2-dependent transcription remains largely unclear. This review aims to cover recent discoveries of the mechanism of transcriptional activation by MLL fusion proteins and to introduce novel roles of SL1 in RNAP2-dependent transcription by discussing how the RNAP1 machinery may be involved in RNAP2-dependent gene regulation.

HMGB1 Interacts with the MLL-AF4 Fusion Complex to Regulate Pro-Leukemic Gene Transcription in Infant Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) in infants carries a poor prognosis and is characterized by cytogenetic rearrangements producing abnormal MLL fusion genes. Clinically effective targeting of the MLL fusion heterocomplex remains challenging, and therapeutic options remain limited. We have observed that the reduced isoform of HMGB1, a chromatin architectural protein that stabilizes DNA and facilitates transcription, is selectively over-expressed in the nuclei of infant MLL-ALL cells. In this study, we generated an HMGB1 siRNA knockdown in primary MLL-ALL cells from 3 infants to test our hypothesis that HMGB1-MLL interactions regulate pro-leukemic gene expression and represent a rational therapeutic target.CD19-selected leukemic blasts were isolated from the cryopreserved bone marrow or peripheral blood specimens of 3 infants with cytogenetically confirmed MLL-AF4 rearrangements. HMGB1 knockdown was confirmed by comparing HMGB1 mRNA and protein expression, by qPCR and Western Blot, in cells transfected with HMGB1 vs. control sequence siRNA. First, determined whether HMGB1 knockdown affected expression of the MLL fusion gene itself, by comparing MLL-AF4 mRNA and protein levels 72 hours after siRNA transfection. HMGB1 knockdown produced a 2.8 (± 0.55)- fold decrease in MLL-AF4 mRNA expression by qPCR (p<0.05), with a corresponding decrease in MLL-AF4 fusion protein expression by Western Blot, in each of the 3 specimens.Next, we determined whether HMGB1 binds functionally relevant regions of the MLL gene. We developed an electrophoretic mobility assay (EMSA) to compare the mobility of lysates from control vs. HMGB1 siRNA-treated infant MLL-ALL cells when mixed with biotinylated oligonucleotides spanning the transcriptionally active domains of MLL1. In each of 3 primary infant MLL-ALL cells, we detected a consistent gel-shift pattern on SDS-PAGE, in wild-type and control siRNA lysates, with oligonucleotides spanning exons 6-9- where many MLL-AF4 fusions occur. The gel-shift was completely abrogated in HMGB1 siRNA lysates. We then compared the expression of MLL target genes involved in leukemic transformation, by qPCR, in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. We observed a significant (p<0.01) reduction in expression of MEIS1 (5.8 ± 2.2-fold decrease), HOXA7 (4.3 ± 0.4-fold decrease) and HOXA9 (3.7 ± 1.5-fold decrease) in infant MLL-ALL cells treated with HMGB1 vs. control siRNA. These data confirmed a role for HMGB1 in MLL gene/target gene regulation at the DNA level.Finally, we considered whether HMGB1, as a scaffold protein, could interact directly with the MLL fusion heterocomplex at the protein level. We immunoprecipitated HMGB1 from the nuclear fraction of wild-type primary infant MLL-ALL cells (n=3 patients), then probed the pull-down for N-terminal MLL (MLLn), C-terminal MLL (MLLc), the MLLn-AF4 fusion, the MLLn-ENL fusion, and the MLL-associated histone 3 methyltransferase DOT1L. MLLn and MLLn-AF4 were strongly detected in all HMGB1 immunoprecipitates. Individual and sequential co-immunoprecipitation of HMGB1 with MLL-AF4 and DOT1L in revealed loss of known complex formation between MLL-AF4 and DOT1L following HMGB1 knockdown. This was accompanied by a 3.4 (± 0.9)-fold decrease in DOT1L mRNA expression (p<0.001) by qPCR and a complete loss of histone 3k79me2 protein expression by Western blot.Taken together, these data suggest a central role for the fully reduced isoform of HMGB1, found in high abundance in infant ALL nuclei, in the formation of the MLL-AF4 transcription complex- including for the stable recruitment of DOT1L and H3K79me2, and in the regulation of MLL target genes such as HOXA9 and MEIS1. We are currently conducting chromatin immunoprecipitation and sequencing studies to identify methylation marks, particularly at H3K79me2, impacted by HMGB1 knockdown in infant ALL cells. We hope these studies will directly inform the development of small molecule inhibitors that specifically disrupt the binding sites and capacities of HMGB1 with MLL, which could synergize with the effects of methyltransferase inhibitors to more completely silence leukemic gene expression in infant ALL and improve the prognosis of this devastating disease. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of DOT1L and PRMT5 Promotes Anti-Tumor Activity in a Human MLL Leukemia Model Induced By CRISPR/Cas9

MLL rearrangements play a crucial role in leukemogenesis. They are associated with a poor prognosis and new treatment strategies are urgently needed. Here, we used the CRISPR/Cas9 system to generate an innovative leukemia model based on 100% pure MLL-AF4/-AF9 rearranged cells derived from umbilical cord blood with indefinite growth in cell culture systems. Our model shared phenotypical, morphological and molecular features of patient leukemic cells faithfully mimicking the nature of the disease. Thus, it serves as ideal fundamental basis for pharmacological studies.The catalytic activity of disruptor of telomeric silencing 1-like histone methyltransferase (DOT1L) is critical for the transforming potential of MLL oncogenes. Therefore, inhibition of DOT1L deals as one specific therapeutic approach for MLL-rearranged leukemias currently tested in clinical trials. However, the previous success was limited by restricted response warranting further investigation of DOT1L combinations. Recently, it has been shown that the inhibition of protein arginine methyltransferase 5 (PRMT5) exhibits anti-tumoral activity against human cell lines in vitro and in MLL mouse models. Here, we used DOT1L (EPZ004777) and PRMT5 (EPZ015666) inhibitors alone or in combination in our human MLL-rearranged model demonstrating a dose-dependent reduced proliferation, impairment of cell cycle, upregulation of differentiation marker CD14 and apoptosis measured by flow cytometry and downregulation of known MLL target genes determined by qPCR. Strikingly, the combination of both compounds led to complementary anti-tumor effects. Our study provides a strong rationale for novel targeted combination therapies to improve the outcome of MLL-rearranged leukemias. DisclosuresNo relevant conflicts of interest to declare.

Peptidomimetics for Targeting Protein-Protein Interactions between DOT1L and MLL Oncofusion Proteins AF9 and ENL.

MLL-fusion proteins, AF9 and ENL, play an essential role in the recruitment of DOT1L and the H3K79 hypermethylation of MLL target genes, which is pivotal for leukemogenesis. Blocking these interactions may represent a novel therapeutic approach for MLL-rearranged leukemia. Based on the 7 mer DOT1L peptide, a class of peptidomimetics was designed. Compound 21 with modified middle residues, achieved significantly improved binding affinities to AF9 and ENL, with KD values of 15 nM and 57 nM, respectively. Importantly, 21 recognizes and binds to the cellular AF9 protein and effectively inhibits the AF9-DOT1L interactions in cells. Modifications of the N- and C-termini of 21 resulted in 28 with 2-fold improved binding affinity to AF9 and much decreased peptidic characteristics. Our study provides a proof-of-concept for development of nonpeptidic compounds to inhibit DOT1L activity by targeting its recruitment and the interactions between DOT1L and MLL-oncofusion proteins AF9 and ENL.

MLL leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing

AbstractGenome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here, we used transcription activator-like effector nucleases to generate t(9;11) chromosomal translocations encoding MLL-AF9 and reciprocal AF9-MLL fusion products in CD34+ human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated MLL target gene expression, high surface CD9 expression, and increased colony-forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient MLL-rearranged leukemias. No secondary pathogenic mutations were revealed by targeted exome sequencing and whole genome RNA-sequencing analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute MLL-rearranged leukemia in vivo, reflecting the genetic simplicity of this disease, and provides an experimental platform for biological and disease-modeling applications.

Final Report of Phase 1 Study of the DOT1L Inhibitor, Pinometostat (EPZ-5676), in Children with Relapsed or Refractory MLL-r Acute Leukemia

Introduction: MLL-rearranged (MLL-r) acute leukemia in children is characterized by young age at presentation and a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) recruit another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of histone H3 lysine 79 (H3K79) at MLL target genes. This results in enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and has been established as a key mechanism for leukemogenesis in MLL-r leukemias (Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Treatment of MLL-rearranged cells and xenograft models with pinometostat led to reduced histone 3 lysine 79 methylation (H3K79me2), decreased MLL target gene expression and selective leukemia cell kill (Daigle, 2013). Here we report the final results of the pinometostat phase 1 trial in children with relapsed/refractory (R/R) MLL-r acute leukemia.Methods: An open label dose escalation study of pinometostat was performed in patients (pts) aged 3 months to 18 years (yr) with R/R MLL-r leukemia (NCT02141828). Pinometostat was administered via continuous intravenous infusion (CIV) until disease progression or unacceptable toxicity. Pts were assigned to one of two aged-based dose escalation schemas developed from simulations of pediatric exposures using a previously reported physiologically-based PK (PBPK) model (Waters, 2014). All patients underwent serial collection of PK and peripheral blood mononuclear cells (PBMC). Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for H3K79me2 levels by ChIP-Seq.Results: 18 pts were enrolled on study with 9 pts dosed at 70 mg/m2/day and 7 pts at 90 mg/m2/day in the older age cohort (1 to 18 yr) plus 2 pts dosed at 45 mg/m2/day in the younger age cohort (<1 yr).Grade ≥3 treatment-emergent adverse events (TEAEs) regardless of attribution reported in >20% of pts were: febrile neutropenia; anemia; leukopenia; thrombocytopenia; hypokalemia; respiratory failure; lymphopenia; neutropenia. Drug-related TEAEs reported in >15% of pts were: anemia; thrombocytopenia; leukopenia; rash; lymphopenia; hypocalcemia; hypophosphatemia; neutropenia; ALT elevation; nausea; vomiting.Dose-limiting toxicities (DLT) were: apnea (70 mg/m2); elevated transaminases (2 at 90 mg/m2) in the >1 year of age cohort, thus defining 70 mg/m2 as the recommended phase 2 dose (RP2D) in older pts. A RP2D was not determined in pts < 1 year of age. Median duration of treatment was 23 days (range 7- 53 days). Pinometostat induced transient decreases in peripheral or marrow leukemic blasts in 7/18 pts, however, these reductions did not meet formal thresholds for objective response.Adjusted mean plasma pinometostat concentrations during the infusion in children >1 yr at 70 and 90 mg/m2/d doses (1151 ng/mL) was comparable to mean steady-state concentrations observed in adult patients at the 80 mg/m2/d and 90 mg/m2/d doses (1320 ng/mL and 1410 ng/mL, respectively) and was within the range of 1000-1600 ng/mL at 90 mg/m2/d in ≥ 1 yr predicted by earlier PBPK modeling results. CSF concentrations of pinometostat were below the lower limit of quantification of 1 ng/mL (n = 8) or very low (< 12 ng/mL) (n = 4), suggesting negligible CSF exposure. H3K79me2 ChIP-Seq on leukemic blasts demonstrated that pinometostat induced reductions in methylation at MLL-r target genes (e.g. HOXA9 and MEIS1) of ≥ 80 % at all post dose time points (15 and 28 days) and doses (70 & 90 mg/m2) tested consistent with DOT1L inhibition.Conclusions: In pediatric pts with R/R MLL-r pinometostat has an acceptable safety profile with a RP2D defined as 70 mg/m2 CIV in children > 1 yr. Pinometostat dose/exposure relationships were comparable between adults and children > 1 yr. Pharmacodynamic evidence of DOT1L inhibition was observed in leukemic blasts. Transient reductions in peripheral or bone marrow blasts were detected in ~40 % of pts, however no objective responses were observed. Based on the biological activity observed and evidence of combination benefit of pinometostat with standard of care and novel agents in preclinical MLL-r models (Daigle 2015 and Klaus 2015) further clinical investigation of pinometostat combinations is warranted. [Display omitted] DisclosuresO'Brien:Seattle Genetics: Research Funding; Celgene: Other: travel expenses for required site investigator meeting, October 2015;; steering committee member for pediatric AML trial, Research Funding; Epizyme: Research Funding; Amgen: Other: participated in one pediatric advisory board for blinatumomab in May 2015, paid consultant fee and travel expenses, Research Funding. Blakemore:Epizyme: Employment. Daigle:Epizyme: Employment. Suttle:Epizyme: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy. Ho:Epizyme: Employment, Equity Ownership.

NPM1 Mutation Contributes to Hematological Dysfunction By Disrupting H3K79 Methylation

NPM1 is one of the most frequent acquired mutated genes in acute myeloid leukemia (AML). Previous studies have shown that NPM1 mutation (NPMc+) established the distinctive gene expression signatures, which were associated with mixed lineage leukemia (MLL)-target genes, like MEIS1 and HOXA cluster. In AML carrying MLL fusion-oncoproteins, DOT1L-mediated histone 3 lysine 79 (H3K79) methylation is implicated in the regulation of MLL-target genes. Compared with MLL abnormalities, NPM1 variants preserve the similar transcriptional characteristics. However, whether NPM1 mutation could affect the histone modification of H3K79 methylation is unknown. In this study, we showed that NPM1 mutation dysregulated the homeostasis of hematopoietic stem and progenitor cells and resulted in ageing-related myeloproliferation in NPMc+ transgenic mouse model. Interestingly, through scanning the chromatin modification related gene profiling, di- and tri- methylated H3K79 were significantly elevated in bone marrow (BM) Lin-Sca-1+c-Kit+ cells (LSKs) of NPMc+ mice comparing to wild type (WT). Meanwhile, in the leukemia cell lines and AML primary BM samples, we confirmed that NPM1 mutated cells expressed the higher level of H3K79 methylation. In vitro assays also indicated that the decrease or increase of methylated H3K79 could be regulated respectively by knockdown or overexpression of NPM1 mutant but not WT. Importantly, with DOT1L inhibitor treatment, reduced di- and tri- methylated H3K79 was observed in OCI-AML3 (NPMc+) strains but not OCI-AML2 (NPM1 WT) cells. In contrast with OCI-AML2, DOT1L inhibitor significantly promoted the cell apoptosis and restrained the cell cycle of OCI-AML3. Moreover, by the means of murine BM colony formation assay, DOT1L inhibitor obviously weakened myeloid cell proliferation in NPMc+ mice, while colony number in WT group did not change. Also, leukemia development was repressed in OCI-AML3-xenografted NOD/SCID mice with the treatment of DOT1L inhibitor. Taken together, NPM1 mutation contributes to hematological dysfunction by disrupting H3K79 methylation, which could be largely attenuated by DOT1L inhibitor. DisclosuresNo relevant conflicts of interest to declare.

Preliminary Report of the Phase 1 Study of the DOT1L Inhibitor, Pinometostat, EPZ-5676, in Children with Relapsed or Refractory MLL-r Acute Leukemia: Safety, Exposure and Target Inhibition

Introduction: MLL-rearranged (MLL-r) acute leukemia in children is characterized by young age at presentation and a poor overall prognosis despite multi-agent chemotherapy. Aberrant fusion proteins involving the MLL histone methyltransferase (HMT) recruit another HMT, DOT1L, to a multi-protein complex leading to aberrant methylation of histone H3 lysine 79 (H3K79) at MLL target genes. This results in enhanced expression of critical genes for hematopoietic differentiation, including HOXA9 and MEIS1, and has been established as a key mechanism for leukemogenesis in MLL-r leukemias (Krivstov, 2007). Pinometostat is a small molecule inhibitor of DOT1L with sub-nanomolar affinity and >37,000 fold selectivity against non-MLL HMTs. Treatment of MLL-rearranged cells and xenograft models with pinometostat led to reduced histone 3, lysine 79 (H3K79) methylation, decreased MLL target gene expression and selective leukemia cell kill (Daigle, 2013). We report the preliminary safety, pharmacokinetics (PK), pharmacodynamics (PD) and target inhibition in leukemia cells of pinometostat in a phase 1 trial in children with relapsed/refractory (R/R) MLL-r acute leukemia.Methods: An open label dose escalation study of pinometostat was performed in patients (pts) aged 3 months to 18 years (yr) with R/R MLL-r leukemia (NCT02141828). Pinometostat was administered via continuous intravenous infusion until disease progression or unacceptable toxicity. Pts were assigned to one of two aged-based dose escalation schemas developed from simulations of pediatric exposures using a previously reported physiologically-based PK (PBPK) model (Waters, 2014). All patients underwent serial collection of PK and peripheral blood mononuclear cells (PBMC) for PD. Leukemic blasts were isolated from PBMCs using flow cytometry and quantified for di-methylation of H3K79 (H3K79-me2) by ChIP-Seq.Results: As of 28-June-2015, 11 pts have enrolled in the dose escalation of the study consisting of 10 pts (6 pts at 70 mg/m2/day, 4 pts at 90 mg/m2/day) in the older age cohort (1 to 18 yr) and 1 pt (45 mg/m2/day) in the younger age cohort (<1 yr).Table 1Patient Characteristicsn (%)Median age, yrs (range)4 (0.33 - 15)Sex (M / F)6/5DiagnosisAML5 (45)ALL4 (36)MLL2 (18)# of prior therapeutic regimens1 - 24 (36)3 - 76 (54)8 - 100>101 (9)Prior allogeneic hematopoietic cell transplant6 (54)Adverse events (AEs) reported in >3 pts regardless of attribution were: anemia, febrile neutropenia, abdominal pain, diarrhea, nausea, vomiting, pain, grade 1 prolonged QTcF, lymphopenia, thrombocytopenia, leukopenia, hypocalcemia, hypokalemia, hypophosphatemia, pleural effusion, respiratory failure, dry skin, rash and hypertension. Grade ≥3 related non-hematologic toxicities include: apnea (n=1 and the only protocol defined dose limiting toxicity), organizing pneumonia (n=1), anorexia (n=1), and febrile neutropenia (n=1). The median duration of treatment was 26 days (range 7- 53 days). Updated response data will be provided.Steady-state plasma concentrations (Css) of pinometostat in children >1 yr at 70 and 90 mg/m2 doses were comparable to that observed in adult patients at equivalent doses and were in the range of 800 - 1600 ng/mL, corroborating earlier PBPK modeling results (projected Css range of 1000-1600 ng/mL at 90 mg/m2/d in ≥1 yr). Steady-state CSF concentrations of pinometostat were low (<2 ng/mL). H3K79-me2 ChIP-Seq demonstrated pinometostat induced reductions in methylation at MLL -r target genes HOXA9 and MEIS1 (range of inhibition = 44-72%)in 2 of 2 pts analyzed to date. Inhibition of H3K79-me2 in leukemic blasts is consistent with DOT1L suppression and additional PK/PD relationships are under investigation.Conclusions: Pinometostat in children with R/R leukemia has an acceptable safety profile. Exposures of pinometostat in children were consistent with PBPK modeling based on adult exposure data, suggesting that dose/exposure relationships are similar between adults and children > 1 yr. Analysis of H3K79-me2 ChIP-Seq data demonstrated PD reductions in methylation of MLL-r target genes expected from DOT1L inhibition. Enrollment and dose escalation continue. DisclosuresO'Brien:Seattle Genetics, Inc.: Research Funding. Pauly:Seattle Genetics, Inc.: Research Funding. Whitlock:Amgen: Honoraria. Thomson:Epizyme, Inc: Employment. Blakemore:Epizyme: Employment. Daigle:Epizyme, Inc: Employment. Pimentel:Epizyme, Inc: Employment. Waters:Epizyme, Inc: Employment. Armstrong:Epizyme, Inc: Consultancy. Ho:Epizyme, Inc: Employment.

The H3K4-Methyl Epigenome Regulates Leukemia Stem Cell Oncogenic Potential

The genetic programs that maintain leukemia stem cell (LSC) self-renewal and oncogenic potential have been well defined; however, the comprehensive epigenetic landscape that sustains LSC cellular identity and functionality is less well established. We report that LSCs in MLL-associated leukemia reside in an epigenetic state of relative genome-wide high-level H3K4me3 and low-level H3K79me2. LSC differentiation is associated with reversal of these broad epigenetic profiles, with concomitant downregulation of crucial MLL target genes and the LSC maintenance transcriptional program that is driven by the loss of H3K4me3, but not H3K79me2. The H3K4-specific demethylase KDM5B negatively regulates leukemogenesis in murine and human MLL-rearranged AML cells, demonstrating a crucial role for the H3K4 global methylome in determining LSC fate.