MLL-fusion Leukemia Research Articles

Prioritization of Eleven-Nineteen-Leukemia Inhibitors as Orally Available Drug Candidates for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is the second most prevalent and fatal form of leukemia. The growth of AML cells harboring oncogenic MLL rearrangements relies on the YEATS domain-containing protein ENL. Many small molecule inhibitors targeting ENL have been developed. To prioritize these inhibitors for in vivo studies, a NanoBRET system was introduced to evaluate their cellular permeability and potency. This screening identified inhibitor 13 as a promising candidate. This inhibitor has remarkable metabolic stability and potent antiproliferative effects on MLL-fusion leukemia cell lines. In AML-xenografted mice, inhibitor 13 significantly improved survival. Subsequent optimization efforts led to the development of SR-C-107 (R), which exhibited strong activity against AML both at the cellular level (CC50(MOLM-13): 1.25 ± 0.18 μM; CC50(MV4-11): 0.81 ± 0.15 μM) and in vivo. These findings establish SR-C-107 (R) as a compelling candidate for AML treatment and lay the groundwork for the development of next-generation AML inhibitors.

IMPDH inhibition activates TLR-VCAM1 pathway and suppresses the development of MLL-fusion leukemia.

Inosine monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in de novo guanine nucleotide synthesis pathway. Although IMPDH inhibitors are widely used as effective immunosuppressants, their antitumor effects have not been proven in the clinical setting. Here, we found that acute myeloid leukemias (AMLs) with MLL-fusions are susceptible to IMPDH inhibitors invitro. We also showed that alternate-day administration of IMPDH inhibitors suppressed the development of MLL-AF9-driven AML invivo without having a devastating effect on immune function. Mechanistically, IMPDH inhibition induced overactivation of Toll-like receptor (TLR)-TRAF6-NF-κB signaling and upregulation of an adhesion molecule VCAM1, which contribute to the antileukemia effect of IMPDH inhibitors. Consequently, combined treatment with IMPDH inhibitors and the TLR1/2 agonist effectively inhibited the development of MLL-fusion AML. These findings provide a rational basis for clinical testing of IMPDH inhibitors against MLL-fusion AMLs and potentially other aggressive tumors with active TLR signaling.

DOT1L O-GlcNAcylation promotes its protein stability and MLL-fusion leukemia cell proliferation.

Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.

Mutant ASXL1 Cooperates with BAP1 to Promote Myeloid Leukemogenesis

ASXL1 mutations occur frequently in myeloid neoplasms, but the mechanisms by which the mutant ASXL1 induces leukemogenesis remain unclear. In the present study, we found a mutually reinforcing effect between a C-terminally truncating ASXL1 mutant (ASXL1-MT) and the histone H2A deubiquitinase BAP1. Biochemical analyses revealed that BAP1 stabilized ASXL1-MT and induced its monoubiquitination at lysine 351. The monoubiquitinated ASXL1-MT in turn increased the nuclear retention and catalytic function of BAP1. Thus, ASXL1-MT forms a hyperactive complex with BAP1 and reduces global ubiquitination of histone H2A at lysine 119 (H2AK119ub) in cells.We assessed the role of the ASXL1-MT/BAP1 complex in hematopoietic differentiation using in vitro cell culture assays with cell lines (32Dcl3 and TF1), mouse bone marrow (BM) cells, and human cord blood (CB) CD34+ cells. These assays revealed that ASXL1-MT alone modestly, and coexpression of ASXL1-MT and BAP1 dramatically impaired multi-lineage differentiation of hematopoietic stem/progenitor cells towards granulocytes, macrophages, mast cells, and mature erythroid cells. Interestingly, ASXL1-MT/BAP1 complex did not inhibit, but rather promoted monocytic differentiation, which may account for the frequent detection of ASXL1 mutations in patients with chronic myelomonocytic leukemia (CMML).We next assessed the role of ASXL1-MT and BAP1 in myeloid leukemogenesis. Coexpression of ASXL1-MT and BAP1 increased the colony forming activity of mouse BM cells, but was not sufficient to transform them. Next, we examined the possible cooperation between ASXL1-MT and RUNX1-ETO in promoting myeloid leukemogenesis because ASXL1 mutations have been frequently detected in RUNX1-ETO leukemia. Forced expression of ASXL1-MT accelerated the development of leukemia driven by RUNX1-ETO9a (a shorter isoform of RUNX1-ETO with a strong leukemogenic activity) in a mouse transplantation model, and also promoted the growth of RUNX1-ETO-expressing human CB cells in vitro . These growth-promoting effects of ASXL1-MT were further enhanced by BAP1 coexpression in RUNX1-ETO leukemia cells. Thus, ASXL1-MT and BAP1 promote myeloid leukemogenesis in cooperation with RUNX1-ETO.Mechanistically, we identified HOXA cluster genes and IRF8 as direct targets of ASXL1-MT and BAP1. RNA-seq and ChIP-qPCR assays revealed that ASXL1-MT/BAP1 complex bound the promoter regions of Hoxa5, Hoxa7, Hoxa9, and Irf8 genes, and induced upregulation of these genes via profound reduction of H2AK119ub. The upregulation of HOXA genes and IRF8 likely contributes to the ASXL1-MT/BAP1-induced leukemogenesis and monopoiesis, respectively.Finally, we deleted BAP1 using CRISPR/Cas9 in mouse BM cells transformed by combined expression of ASXL1-MT and a SETBP1 mutant (SETBP1-D868N). Strikingly, BAP1 depletion in the cells led to a significant decrease of leukemogenicity and Hoxa gene expression. We then examined whether Bap1 is also required for leukemogenicity of MLL-fusion leukemia, a well-known leukemia subtype characterized by Hox gene dysregulation. Again, Bap1 depletion reduced colonogenicity and Hoxa gene expression in MLL-AF9-expressing mouse BM cells. Moreover, we confirmed the essential role of endogenous BAP1 to support the growth of human myeloid leukemia cell lines with ASXL1 mutations (Kasumi-1, MEG-01 and TS9;22) or MLL-fusions (MOLM13 and THP1).In summary, our study demonstrated the critical role of BAP1 in ASXL1-MT-induced aberrant myeloid differentiation, myeloid leukemogenesis, and upregulation of HOXA genes and IRF8 . Targeting enzymatic activity of BAP1 can be a promising therapeutic strategy for myeloid neoplasms with ASXL1 mutations, and potentially for a broad range of myeloid neoplasms with HOX dysregulation. DisclosuresKitamura:Daiichi Sankyo: Research Funding.

Discovery of DDO-2213 as a Potent and Orally Bioavailable Inhibitor of the WDR5-Mixed Lineage Leukemia 1 Protein-Protein Interaction for the Treatment of MLL Fusion Leukemia.

WD repeat-containing protein 5 (WDR5) is essential for the stability and methyltransferase activity of the mixed lineage leukemia 1 (MLL1) complex. Dysregulation of the MLL1 gene is associated with human acute leukemias, and the direct disruption of the WDR5-MLL1 protein-protein interaction (PPI) is emerging as an alternative strategy for MLL-rearranged cancers. Here, we represent a new aniline pyrimidine scaffold for WDR5-MLL1 inhibitors. A comprehensive structure-activity analysis identified a potent inhibitor 63 (DDO-2213), with an IC50 of 29 nM in a competitive fluorescence polarization assay and a Kd value of 72.9 nM for the WDR5 protein. Compound 63 selectively inhibited MLL histone methyltransferase activity and the proliferation of MLL translocation-harboring cells. Furthermore, 63 displayed good pharmacokinetic properties and suppressed the growth of MV4-11 xenograft tumors in mice after oral administration, first verifying the in vivo efficacy of targeting the WDR5-MLL1 PPI by small molecules.

Tip60 activates Hoxa9 and Meis1 expression through acetylation of H2A.Z, promoting MLL-AF10 and MLL-ENL acute myeloid leukemia.

Chromosome translocations involving the MLL gene are common rearrangements in leukemia. Such translocations fuse the MLL 5'-region to partner genes in frame, producing MLL-fusions that cause MLL-related leukemia. MLL-fusions activate transcription of target genes such as HoxA cluster and Meis1, but the underlying mechanisms remain to be fully elucidated. In this study, we discovered that Tip60, a MYST-type histone acetyltransferase, was required for the expression of HoxA cluster and Meis1 genes and the development of MLL-fusion leukemia. Tip60 was recruited by MLL-AF10 and MLL-ENL fusions to the Hoxa9 locus, where it acetylated H2A.Z, thereby promoting Hoxa9 gene expression. Conditional deletion of Tip60 prevented the development of MLL-AF10 and MLL-ENL leukemia, indicating that Tip60 is indispensable for the leukemogenic activity of the MLL-AF10 and MLL-ENL-fusions. Our findings provide novel insight about epigenetic regulation in the development of MLL-AF10 and MLL-ENL-fusion leukemia.

Investigating the Roles of the Yeats Domain in MLL-ENL Mediated Leukemogenesis

Approximately 10% of acute leukemia involves rearrangement at chromosome 11q23, giving rise to a relatively aggressive form of acute leukemia characterized by MLL1 (KMT2A) fusion proteins. Despite the identification of >100 MLL1 fusion partners, the majority are members of several similar transcriptional activation complexes including: The Super Elongation Complex (SEC), AEP and EAP (SEC used hereafter). MLL fusion-driven acute leukemia is characterized by deregulated activity of the SEC and the H3K79 methyltransferase DOT1L. This leads to altered epigenetic landscapes at and deregulated transcription of pro-leukemic MLL1-fusion target genes like HoxA9 and Meis1. Thus, targeting these transcriptional and epigenetic complexes has become an attractive therapeutic strategy for treating MLL-fusion leukemia. Eleven-Nineteen-Leukemia (ENL or MLLT1) is the third most common MLL1 fusion partner and a component of the SEC. Recently, wild type ENL was identified as an essential factor for leukemic cell growth. The ENL protein possesses a C-terminal ANC-homology domain (AHD) necessary for SEC recruitment and is essential for MLL-fusion mediated leukemogenesis. In addition, ENL contains a highly conserved N-terminal YEATS domain that functions as an epigenetic reader for acetylated H3K9, H3K18 or H3K27, which is essential for leukemic cell growth. Additionally, the ENL YEATS domain directly interacts with the Polymerase Associated Factor 1 complex (PAF1c), an epigenetic regulator protein complex essential for MLL-fusion mediated leukemogenesis. These studies highlight the importance of the YEATS domain in regulating wild type ENL function in leukemic cells. However, the importance of the YEATS domain in the context of MLL-ENL mediated leukemia remains to be elucidated. In this study, we investigate the clinical relevance and leukemic importance of the ENL YEATS domain in MLL-ENL leukemias. We first analyzed t(11;19) (MLL-ENL) patient data to determine the sites of chromosomal translocation within the ENL gene. We found that the YEATS domain (coded by exons 2 through 4) is retained in 84.1% of MLL-ENL patients (n=302). Specifically, 50.7% (n=153) of these patients possess breakpoints located 5' of the first exon of the ENL gene, while 33.4% (n=101) of the patients display breakpoints within the first intron of ENL gene. These data point towards a tendency for YEATS domain retention in MLL-ENL fusion proteins in t(11;19) patients. We next tested whether the YEATS domain was functional in MLL-ENL mouse leukemia models. Our data shows the YEATS domain is required for MLL-ENL leukemogenesis in vivo, as deletion of the YEATS domain destroys MLL-ENL leukemogenesis and increases apoptosis in cell culture. Transcriptionally, deletion of the YEATS domain decreased expression of pro-leukemic genes such as Meis1 and the anti-apoptotic gene Bclxl. To dissect the contribution of different YEATS domain functions in MLL-ENL leukemogenesis, we engineered YEATS domain mutants defective in interacting with PAF1 or acetylated H3K9/K18/K27. Disrupting the YEATS-PAF1 or YEATS-H3Kac interaction decreased MLL-ENL mediated colony formation exvivo and significantly increased leukemia latency in vivo. The MLL-ENL YEATS domain mutants will be used in future studies to determine how the YEATS domain affects 1) MLL-ENL fusion localization, 2) key protein complexes localization (i.e. SEC and PAF1c) and 3) the epigenetic landscapes (i.e. H3K79me2/3 and H3K4me3) at pro-leukemic targets. To further interrogate the YEATS-PAF1 interaction in MLL-ENL mediated leukemia, we identified the minimal region of the PAF1 protein required for the YEATS-PAF1 interaction. This PAF1 protein fragment will be used to biochemically characterize the structure of the PAF1-YEATS interaction, which might aid in therapeutically targeting specific YEATS interactions in MLL-ENL leukemia. Our results demonstrate for the first time, to our knowledge, an essential role for the YEATS domain in MLL-ENL mediated leukemogenesis. Additionally, our genetic studies elucidate the importance of the YEATS domain interaction with either the PAF1c or H3Kac in MLL-ENL leukemias. Taken together, our study establishes a rationale for exploring the effectiveness of small molecule development aimed at disrupting either the YEATS-H3Kac or the YEATS-PAF1 interaction as a therapeutic intervention for treating MLL-ENL leukemia patients. Disclosures No relevant conflicts of interest to declare.

BCOR Binding to MLL-AF9 Is Essential for Leukemia via Altered EYA1, SIX, and MYC Activity.

MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population.

SETDB1 Mediated H3K9 Methylation Suppresses MLL-Fusion Target Expression and Leukemic Transformation

Genes encoding epigenetic proteins are mutated in about 70% of Acute Myeloid Leukemia patients underscoring their role in leukemic transformation. Epigenetic deregulation of the HOXA gene cluster drives transformation of about 50% of acute myeloid leukemia (AML), such as those harboring MLL rearrangements, NPM mutations and others. Indeed, expression of Hoxa9 and its co-factor Meis1 is sufficient to transform bone marrow cells into a lethal AML. We have shown that epigenetic regulation of Hoxa9 and Meis1 is mediated through the Polymerase Associated Factor 1 complex (PAF1c). The PAF1c binds to RNA polymerase II and recruits enzymes that alter epigenetic landscapes and influence gene transcription. We and others have shown that the PAF1c directly binds and recruits MLL and MLL fusion proteins to target genes such as Hoxa9 and Meis1 to deliver H3K4 methylation or H3K79 methylation activity respectively. Importantly, the PAF1c-MLL1 interaction is necessary for the growth of MLL-fusion leukemia cells but appears dispensable for normal hematopoietic stem and progenitor cells (HSPCs) function. This suggests the PAF1c is differentially required in leukemic cells compared to its normal counterparts. Thus, we aimed to 1) define the role of the PAF1c in normal hematopoiesis, and 2) identify mechanisms regulating PAF1c function that may be deregulated in leukemia. To understand the role of the PAF1c in hematopoiesis, we conditionally deleted the PAF1c subunit Cdc73 in HSPCs in hematopoietic cells. Conditional hematopoietic deletion of Cdc73 leads to lethality within a week due to a depletion of c-kit+ cells. Characterization revealed a cell autonomous requirement for Cdc73 to prevent apoptosis in HSPCs. Gene expression profiling on both c-kit+ hematopoietic cells and AML cells revealed that Hoxa9/Meis1 gene programs are differentially responsive to loss of Cdc73. This work revealed an essential role for the PAF1c subunit in maintaining viability of HSPCs and points to AML specific functions for the PAF1c. Thus, we utilized immunoprecipitation followed by mass spectroscopy to identify novel PAF1c interactions in AML cells. We found a novel interaction with several H3K9 methyltransferases including SETDB1. We demonstrate that the PAF1c-SETDB1 interaction reduces expression of Hoxa9 and Meis1 by promoting deposition of H3K9me3. These findings mirrored human AML patient samples that showed SETDB1 expression was inversely correlated with HOXA9 and MEIS1 expression. Further, SETDB1 expression is correlated to disease status and overall survival in AML patients. We recapitulated these findings in mice, where high expression of SETDB1 delayed MLL-AF9 mediated disease progression by promoting differentiation of AML cells. We also explored the biological impact of treating normal and malignant hematopoietic cells with an H3K9 methyltransferase inhibitor, UNC0638. While AML cells demonstrate cytotoxicity to UNC0638 treatment, normal bone marrow cells exhibit an expansion of HSPCs. Consistent with these data, we show that bone marrow treated with UNC0638 is more amenable to transformation by MLL-AF9. Next generation sequencing of AML cells shows that high expression of SETDB1 induces repressive changes to the promoter epigenome and downregulation of genes linked with AML, including Dock1 and the MLL-AF9 target genes Hoxa9, Six1, and others. These data reveal novel targets of SETDB1 in AML that point to a role for SETDB1 in negatively regulating pro-leukemic target genes and suppressing AML. Disclosures Maillard: Genentech: Consultancy; Regeneron: Consultancy.

Meningioma 1 is indispensable for mixed lineage leukemia-rearranged acute myeloid leukemia.

Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL-r) are one of the most frequent chromosomal aberrations in acute myeloid leukemia. We evaluated the function of Meningioma 1 (MN1), a co-factor of HOXA9 and MEIS1, in human and murine MLL-rearranged leukemia by CRISPR-Cas9 mediated deletion of MN1. MN1 was required for in vivo leukemogenicity of MLL positive murine and human leukemia cells. Loss of MN1 inhibited cell cycle and proliferation, promoted apoptosis and induced differentiation of MLL-rearranged cells. Expression analysis and chromatin immunoprecipitation with sequencing from previously reported data sets demonstrated that MN1 primarily maintains active transcription of HOXA9 and HOXA10, which are critical downstream genes of MLL, and their target genes like BCL2, MCL1 and Survivin. Treatment of MLL-rearranged primary leukemia cells with anti-MN1 siRNA significantly reduced their clonogenic potential in contrast to normal CD34+ hematopoietic progenitor cells, suggesting a therapeutic window for MN1 targeting. In summary, our findings demonstrate that MN1 plays an essential role in MLL fusion leukemias and serve as a therapeutic target in MLL-rearranged acute myeloid leukemia.

PF208 MN1 IS INDISPENSABLE FOR MLL‐REARRANGED ACUTE MYELOID LEUKEMIA

Background:Mixed lineage leukemia (MLL/KMT2A) rearrangements (MLL‐r) are one of the most frequent chromosomal aberrations in AML. In addition, Meningioma 1 (MN1) expression serves as a prognostically important factor in AML patients being associated with poor prognosis. Overexpression of MN1 induces AML in mice and causes resistance to cytarabine and all‐trans‐retinoic acid (ATRA) induced cytotoxic/differentiation effects.Aims:To evaluate the function of MN1, a cofactor of HOXA9 and MEIS1, in human and murine AML cells by CRISPR‐Cas9 mediated deletion of MN1.Methods:MN1 was deleted in murine cells transformed by MLL‐AF9/HOXA9/HOXA9MEIS1/E2A‐HLF and 10 human leukemia cell lines (THP‐1, MV4–11, NB4, OCI‐AML2, OCI‐AML3, U937, K562, Kasumi‐1, HL‐60 and HEL). Murine and human leukemic models with engineered deletion of MN1 were studied as single cell clones and were characterized in vitro and in vivo.Results:Out of 96 to 288 CRISPR‐Cas9‐MN1‐transduced single sorted clones per cell line, 30 percent of them proliferated and only 0 to 8.9% of them were found to have a MN1‐deletion. Interestingly, only 1 MN1‐deleted clone was obtained from four different murine cell lines with HOX‐dependent oncogene expression suggesting that MN1 loss impairs clonogenicity of MLL‐r and HOXA9‐transformed cells. Loss of MN1 reduced in vitro proliferation and impaired colony‐forming potential up to 22‐fold in MLL‐r murine and human leukemia cells. MN1 deletion also suppressed cell cycle, promoted apoptosis and induced differentiation in MLL positive leukemia cells. MN1 was critically required for in vivo leukemogenicity of MLL‐AF9 murine leukemic cells. Mice transplanted with MLL‐AF9/Mn1null cells had <1% engraftment in all ten mice, whereas mice transplanted with MLL‐AF9/Mn1 wt cells showed >90% engraftment at 4 weeks with high WBC counts. Loss of Mn1 significantly prolonged survival of mice transplanted with MLL‐AF9/Mn1null cells as compared to mice transplanted with MLL‐AF9/Mn1 wt cells (median survival of 35 days). Additionally, we transplanted MN1 wildtype or MN1null cells of THP‐1 and MV‐4‐11 subcutaneously in NOD‐SCID mice and monitored tumor formation. Deletion of MN1 led to significantly reduced tumor volumes in both THP‐1 and MV‐4‐11 cells (MN1null) as compared to their MN1 wild type control. ChIP sequencing and gene expression analysis suggested that MN1 is required in MLL‐r leukemias to maintain expression of the distal Hoxa cluster and Meis1 and as a cofactor of the Hoxa9/Meis1 transcriptional complex and their target genes essential for leukemogenic activity. Also, we treated CD34+ cells from healthy donors and MLL‐AF9 positive AML cells with control/anti‐MN1 siRNA. MN1 expression was found to be critical for proliferation of MLL‐transformed leukemic cells while it had no effect on CD34+ cells from healthy donors, proposing MN1 as a therapeutic target in MLL‐transformed AML.Summary/Conclusion:Our study offers insight to the function of endogenous MN1 expression in leukemogenesis of MLL‐r AML. Our data shows that MN1 is essential for MLL fusion leukemias via regulation of Hoxa cluster and Meis genes and serves as a therapeutic target in MLL‐r AML.

MLL-Fusion Leukemia Dependence on MBNL1 Is Associated with Alternative Splicing of Oncogenic Proteins

Leukemia is the most common childhood cancer, and while outcomes for most children have improved significantly, the prognosis in infant leukemia remains dire. The majority of infant leukemia, either acute myeloid (AML) or acute lymphoid (ALL), is caused by reciprocal translocations of the MLL-gene. Prior studies show that one of the most consistently overexpressed genes in these leukemias (compared to all other leukemias) is the RNA binding protein muscleblind-like 1 (MBNL1). We found that MBNL1 knockdown significantly impairs propagation of MLL-rearranged (MLLr) leukemic cells in vitro and in vivo using human cell lines and transformed murine cells. To further characterize the role of MBNL1 in acute leukemia, we performed shRNA knockdown experiments in MLLr and non-MLLr leukemia cell lines and in primary patient samples. While MBNL1 knockdown does also impair growth of non-MLLr leukemic cells, the effect is less pronounced. In a 5-day growth experiment MBNL1-knockdown MLLr cells (THP-1) displayed a median 71% reduced growth compared to controls, whereas non-MLLr cells (HL-60) displayed only a median 32% growth reduction (p=0.0001). Cells from two patients with MLLr AML (one with MLL-AF9 and one with MLL-AF10 fusion) underwent shNT (non-targeting) or shMBNL1 transduction.. Unsorted cells were transplanted into NSGS mice. Mice were observed until showing signs of distress and then analyzed for engraftment of human cells and abundance of transduced cells (venus-positive). In the shNT group there was robust persistence of transduced cells (7%-98% of human cells), whereas shMBNL1-transduced cells were not detected or comprised <1% of human cells in most of the recipient mice.Given that MBNL1 is known to regulate alternative splicing, we used unbiased RNAseq along with a novel analytic splice-junction and intron-quantification toolkit (AltAnalyze) to determine splicing changes induced by knockdown of MBNL1 in the MLLr leukemia cell line MOLM-13. In a parallel analysis, we determined splicing differences between MLLr and cytogenetically-normal (CN) AML patient samples. We then compared these two results to determine the splicing events regulated by MBNL1 and assess the contribution of MBNL1 to splicing events observed in primary MLLr leukemias. Strikingly, this comparative analysis found that 88% of overlapping differentially expressed splicing events (75 out of 85) were concordant between patient MLLr and CN-AML as compared to control versus MBNL1 knockdown. The most common class of splicing event that occurred with MBNL1 knockdown was intron retention. Specifically, our findings suggest that MBNL1 knockdown restores intron retention, and that MBNL1 overexpression promotes expression of protein-coding genes that would otherwise be suppressed through intron retention-introduced premature termination codons. Several genes whose transcripts are alternatively spliced by MBNL1 have prior associations with cancer, most notably DOT1L and SETD1A which are specifically implicated in MLLr leukemia. Splicing validation through RT-PCR confirmed increased intron retention in DOT1L and SETD1A transcripts after MBNL1 knockdown. Interestingly, one target of MBNL1 is the MBNL1 mRNA itself, with resultant exclusion of exon 5. MBNL1 lacking exon 5 has a stronger affinity to RNA. In summary, our data suggests that MBNL1 plays a key role in the pathogenesis of MLL-fusion leukemia, wherein it stabilizes the transcripts of multiple leukemogenic genes including DOT1L and SETD1A. Proteins such as DOT1L are critical for transcriptional activation of downstream targets of the MLL-fusion protein (including activation of MBNL1, creating a positive feedback loop). Additionally, high levels of MBNL1 protein may alter splicing in ways that enhance MBNL1 functionality. DisclosuresNo relevant conflicts of interest to declare.

The Role of LAMP5 in Innate Immune Signaling Is Critical for the Survival of MLL Leukemias

IntroductionChromosomal translocations involving the Mixed Lineage Leukemia (MLL, also called KMT2A) gene account for around 80% of the acute lymphoid leukemia (ALL) and 35%-50% of the acute myeloid leukemia (AML) diagnosed in infants, and are associated with poor outcomes. Despite improvements of conventional chemotherapies and treatments, patients with MLL-rearranged leukemia have weak response to treatment and poor prognosis. There is thus an urgent need to understand the molecular pathogenesis of these leukemias to develop more effective therapies. Gene expression studies show that MLL-leukemias uniquely display over-expression of Lysosome-associated Membrane Protein 5 (LAMP5) in both ALL and AML In normal hematopoiesis, LAMP5 expression is restricted to non-activated plasmacytoid dendritic cells (pDC), where it has been shown to be required for transport of TLR9 from VAMP3+/LAMP2+/LAMP1- endolysosomal vesicles to the LAMP1+ late lysosomal compartments, modulating the signaling and production of Type I interferon to TNF production. Here, we tested the hypothesis that LAMP5 is required for MLL leukemia wherein it modulates innate immune signaling.Methods and ResultsIn previous studies, we found LAMP5 expression being restricted to MLL-fusion leukemia cell lines. Additionally, in an inducible model, LAMP5 expression directly correlated with that of the MLL-fusion protein MLL-AF9. . Knockdown on LAMP5 in MLL-fusion leukemia cells inhibited their propagation both in vitro and in vivo whereas the non-MLL leukemias were unaffected. To determine the mechanisms by which LAMP5 promotes growth in MLL leukemias, first we studied the localization of LAMP5 in these cells. Using immunofluorescent confocal microscopy we detected LAMP5 mainly in LAMP2+/LAMP1+ compartments. Additionally, LAMP5 co-localized with MYD88, a known scaffold protein required for IL1R/TLR signaling. Accordingly, LAMP5-knockdown abrogated TLR signaling as evidenced by reduced activation of JNK, MAPK p38, IRF7 and NFKB. Conversely, over-expression of LAMP5 in the non-MLL leukemia cell line Kasumi-1 led to increased activation of JNK, p38, IRF7 and NFKB, indicating that the innate-immune signaling pathway is regulated by LAMP5 expression in leukemias.rConclusionsCollectively, these results demonstrate that LAMP5 is required for the survival of MLL leukemia and that it plays an important role in the activation of the IL1/TLR signaling pathway. Overall, based on our results and the limited expression in normal hematopoiesis, we propose that LAMP5 could potentially serve as a therapeutic target with a wide therapeutic-window to treat MLL-leukemias. DisclosuresNo relevant conflicts of interest to declare.

Mutant ASXL1 cooperates with BAP1 to promote myeloid leukaemogenesis

ASXL1 mutations occur frequently in myeloid neoplasms and are associated with poor prognosis. However, the mechanisms by which mutant ASXL1 induces leukaemogenesis remain unclear. In this study, we report mutually reinforcing effects between a C-terminally truncated form of mutant ASXL1 (ASXL1-MT) and BAP1 in promoting myeloid leukaemogenesis. BAP1 expression results in increased monoubiquitination of ASXL1-MT, which in turn increases the catalytic function of BAP1. This hyperactive ASXL1-MT/BAP1 complex promotes aberrant myeloid differentiation of haematopoietic progenitor cells and accelerates RUNX1-ETO-driven leukaemogenesis. Mechanistically, this complex induces upregulation of posterior HOXA genes and IRF8 through removal of H2AK119 ubiquitination. Importantly, BAP1 depletion inhibits posterior HOXA gene expression and leukaemogenicity of ASXL1-MT-expressing myeloid leukemia cells. Furthermore, BAP1 is also required for the growth of MLL-fusion leukemia cells with posterior HOXA gene dysregulation. These data indicate that BAP1, which has long been considered a tumor suppressor, in fact plays tumor-promoting roles in myeloid neoplasms.

Correction: Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

Correction: Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia

A p53-MDM2 Interaction Inhibitor, DS-5272, Inhibits the Development of MLL-Fusion Leukemia with the Assistance of Tumor Immunity

MLL-fusion leukemia is an aggressive form of leukemia carrying chimeric fusion of the MLL gene. Adverse therapy response of MLL-fusion leukemia is partly associated with the attenuated p53 response. The principal cellular antagonist of p53 is an E3 ubiquitin ligase MDM2. MDM2 binds to p53 and induces proteasomal degradation to downregulate p53 protein level. Therefore, targeting of p53-MDM2 interaction to reactivate p53 function is an attractive therapeutic approach for MLL-fusion leukemia. In this study, we assessed the effect of an orally active inhibitor of p53-MDM2 interaction, DS-5272, in a mouse leukemia model driven by MLL-AF9. DS-5272 inhibited in vitro growth of mouse leukemia cells transformed by MLL-AF9 with the IC50 value in the nanomolar range. A single administration of DS-5272 in vivo upregulated expression of p53 protein as well as its target genes, and induced cell cycle arrest, apoptosis, and differentiation of MLL-AF9 cells. Multiple oral doseadministration of DS-5272 (3 times per week) caused nearly complete tumor regressions of MLL-AF9 leukemia cells that were sustained well beyond the drug administration period with tolerable toxicity. In contrast, DS-5272 showed little effect on p53-deficient bone marrow cells transformed by MLL-AF9 both in vitro and in vivo, confirming that the antileukemic effect of DS-5272 is mediated by p53 activation.Despite the remarkable effect of DS-5272 against MLL-AF9 leukemia with wild-type p53, all mice eventually developed leukemia after a long latency, indicating the existence of leukemia stem cells (LSCs) that are resistant to p53 activation. The therapy-resistant LSCs were relatively enriched within the bone marrow (BM) endosteal region where osteoblasts reside. In contrast, LSCs in the BM central portion were eliminated almost completely by the DS-5272 treatment. Thus, LSCs within the osteoblast-rich area appear to be protected from p53-induced cell death. RNA-Seq analyses revealed the upregulation of inflammation- and interferon-associated genes in MLL-AF9 leukemia cells treated with DS-5272. Furthermore, DS-5272 treatment induced upregulation of PD-L1, a well-known suppressor of tumor immunity, in MLL-AF9 cells. These expression changes suggest that p53 activation triggered an immune-inflammatory response that led to leukemia regression. Interestingly, LSCs reside in the BM endosteal region expressed higher level of PD-L1 compared with those in the BM central portion, which may account for their low sensitivity to DS-5272.To examine the potential contribution of tumor immunity to the DS-5272-mediated suppression of leukemic growth, we next assessed the in vivo effect of DS-5272 on MLL-AF9 leukemia using immunodeficient NOD.Cg- Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice as recipients. Strikingly, the antileukemia effect of DS-5272 was markedly attenuated in NSG mice, indicating the important role of tumor immunity to enhance the efficacy of DS-5272. We then depleted PD-L1 in MLL-AF9 cells using the CRISPR/Cas9 system, and found that PD-L1-depleted MLL-AF9 cells become more sensitive to DS-5272 treatment. These data suggest that DS-5272 inhibits the development of MLL-AF9 leukemia with the assistance of tumor immunity, and its therapeutic efficacy is enhanced by PD-L1 inhibition in MLL-AF9 cells.In summary, our study demonstrated the potent antitumor activity of a p53-MDM2 interaction inhibitor, DS-5272, against MLL-fusion leukemia with intact p53. LSCs reside within the BM endosteal region express high level of PD-L1 and are relatively resistant to DS-5272 treatment. Combining p53-MDM2 inhibitors with the immune checkpoint inhibitors may result in synergistic enhancement of cell death and improved efficacy in the treatment of myeloid leukemia. DisclosuresKitamura:Daiichi Sankyo: Research Funding.

The PAF complex regulation of Prmt5 facilitates the progression and maintenance of MLL fusion leukemia.

Acute myeloid leukemia (AML) is a disease associated with epigenetic dysregulation. 11q23 translocations involving the H3K4 methyltransferase MLL1 (KMT2A) generate oncogenic fusion proteins with deregulated transcriptional potential. The Polymerase Associated Factor complex (PAFc) is an epigenetic co-activator complex that makes direct contact with MLL fusion proteins and is involved in AML, however its functions are not well understood. Here, we explored the transcriptional targets regulated by the PAFc that facilitate leukemia by performing RNA-sequencing after conditional loss of the PAFc subunit Cdc73. We found Cdc73 promotes expression of an early hematopoietic progenitor gene program that prevents differentiation. Among the target genes, we confirmed the protein arginine methyltransferase Prmt5 is a direct target that is positively regulated by a transcriptional unit that includes the PAFc, MLL1, HOXA9 and STAT5 in leukemic cells. We observed reduced PRMT5-mediated H4R3me2s following excision of Cdc73 placing this histone modification downstream of the PAFc and revealing a novel mechanism between the PAFc and Prmt5. Knock down or pharmacologic inhibition of Prmt5 causes a G1 arrest and reduced proliferation resulting in extended leukemic disease latency in vivo. Overall, we demonstrate the PAFc regulates Prmt5 to facilitate leukemic progression and is a potential therapeutic target for AMLs.

The full transforming capacity of MLL-Af4 is interlinked with lymphoid lineage commitment

AbstractChromosome rearrangements involving the mixed-lineage leukemia gene (MLL) create MLL-fusion proteins, which could drive both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The lineage decision of MLL-fusion leukemia is influenced by the fusion partner and microenvironment. To investigate the interplay of fusion proteins and microenvironment in lineage choice, we transplanted human hematopoietic stem and progenitor cells (HSPCs) expressing MLL-AF9 or MLL-Af4 into immunodeficient NSGS mice, which strongly promote myeloid development. Cells expressing MLL-AF9 efficiently developed AML in NSGS mice. In contrast, MLL-Af4 cells, which were fully oncogenic under lymphoid conditions present in NSG mice, displayed compromised transformation capacity in a myeloid microenvironment. MLL-Af4 activated a self-renewal program in a lineage-dependent manner, showing the leukemogenic activity of MLL-Af4 was interlinked with lymphoid lineage commitment. The C-terminal homology domain (CHD) of Af4 was sufficient to confer this linkage. Although the MLL-CHD fusion protein failed to immortalize HSPCs in myeloid conditions in vitro, it could successfully induce ALL in NSG mice. Our data suggest that defective self-renewal ability and leukemogenesis of MLL-Af4 myeloid cells could contribute to the strong B-cell ALL association of MLL-AF4 leukemia observed in the clinic.

Abstract 5489: Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia

Abstract Mixed lineage leukemia (MLL) is an aggressive form of leukemia where the MLL gene is translocated and fused to more than 80 different nuclear, cytoplasmic and membrane proteins. MLL-AF9 is one of the most common MLL-fusions. This fusion partner is known to recruit several multiprotein complexes including the AEP, SEC and DOT1L complexes leading to transcriptional activation. In particular, the recruitment of Disruptor of telomeric silencing 1-like (DOT1L), a H3K79 histone methyltransferase, is essential for leukemogenesis by multiple MLL fusion proteins. We mapped the binding site to a 10 amino acid segment (865 - 874) on DOT1L where AF9 binds. Peptides derived from this region that show dose-dependent disruption of this interaction. We also demonstrated that DOT1L mutants lacking these 10 residues did not support transformation by MLL-AF9. Encouraged by these results, we generated both genetic and chemical tools to elucidate the role of DOT1L recruitment to the MLL fusion partners and the mechanism of leukemogenic inhibition by disrupting the protein-protein interaction (PPI) between MLL-AF9 and DOT1L. Fl/Fl DOT1L MLL-AF9 Cre+ cell lines were generated with different constructs of DOT1L. These constructs consisted of DOT1L mutants lacking the 10 amino acid binding site, a I867A point mutant known to block DOT1L binding, and an enzymatic mutant known to yield a catalytically inactive protein. As control cell lines, MLL-AF6, a MLL-fusion containing a cytoplasmic protein, and E2A-HLF, a non-DOT1L dependent fusion, were generated to demonstrate the specific effects of generated DOT1L mutant constructs. Both DOT1L PPI mutants impaired the transformation by MLL-AF9 and induced cell death by inducing apoptosis and cell cycle arrest similarly to enzymatic inhibition. These results established a foundation for discovering small-molecule inhibitors that disrupting the AF9-DOT1L as potential disease-specific therapies that target chromatin modifications in this highly aggressive leukemia. A high throughput screening was conducted identifying several different chemical classes of small molecules that bind to the AF9 C-terminal hydrophobic binding site and disrupt the PPI between DOT1L and MLL-AF9 fusion protein. Identified small molecule inhibitors were validated with series of biochemical, functional and cell-based assays. Validated compounds selectively inhibit the growth of the DOT1L dependent murine cells and induce cell death in a similar manner to the genetic approach. The small molecules also showed specificity in killing human MLL-fusion cell lines in comparison to non-MLL fusion leukemia. These results show that blocking the recruitment of DOT1L by AF9 using both genetic and chemical tools eliminate MLL-AF9 mediated immortalization emphasizing an essential function for this interaction in leukemogenesis and warrant further development of the identified small-molecule inhibitors. Citation Format: Sierrah M. Grigsby, James Ropa, Justin Serio, Chenxi Shen, Jennifer Chase, Ivan Maillard, Andrew Muntean, Zaneta Nikolovska-Coleska. Using genetic and chemical approaches to probe the mechanism of DOT1L recruitment in MLL fusion leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5489. doi:10.1158/1538-7445.AM2017-5489

Replication Study: Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia.

In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fung et al., 2015), that described how we intended to replicate selected experiments from the paper "Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia" (Dawson et al., 2011). Here, we report the results of those experiments. We found treatment of MLL-fusion leukaemia cells (MV4;11 cell line) with the BET bromodomain inhibitor I-BET151 resulted in selective growth inhibition, whereas treatment of leukaemia cells harboring a different oncogenic driver (K-562 cell line) did not result in selective growth inhibition; this is similar to the findings reported in the original study (Figure 2A and Supplementary Figure 11A,B; Dawson et al., 2011). Further, I-BET151 resulted in a statistically significant decrease in BCL2 expression in MV4;11 cells, but not in K-562 cells; again this is similar to the findings reported in the original study (Figure 3D; Dawson et al., 2011). We did not find a statistically significant difference in survival when testing I-BET151 efficacy in a disseminated xenograft MLL mouse model, whereas the original study reported increased survival in I-BET151 treated mice compared to vehicle control (Figure 4B,D; Dawson et al., 2011). Differences between the original study and this replication attempt, such as different conditioning regimens and I-BET151 doses, are factors that might have influenced the outcome. We also found I-BET151 treatment resulted in a lower median disease burden compared to vehicle control in all tissues analyzed, similar to the example reported in the original study (Supplementary Figure 16A; Dawson et al., 2011). Finally, we report meta-analyses for each result.