Chromosomal rearrangements of the mixed lineage leukemia (MLL/KMT2A) gene leading to oncogenic MLL-fusion proteins occur in ~10% of acute leukemias and are associated with poor clinical outcomes, emphasizing the need for new treatment modalities. Inhibition of the DOT1-like histone H3K79 methyltransferase (DOT1L) is a specific therapeutic approach for such leukemias that is currently being tested in clinical trials. However, in most MLL-rearranged leukemia models responses to DOT1L inhibitors are limited. Here, we performed deep-coverage short hairpin RNA sensitizer screens in DOT1L inhibitor-treated MLL-rearranged leukemia cell lines and discovered that targeting additional nodes of MLL complexes concomitantly with DOT1L inhibition bears great potential for superior therapeutic results. Most notably, combination of a DOT1L inhibitor with an inhibitor of the MLL-Menin interaction markedly enhanced induction of differentiation and cell killing in various MLL disease models including primary leukemia cells, while sparing normal hematopoiesis and leukemias without MLL rearrangements. Gene expression analysis on human and murine leukemic cells revealed that target genes of MLL-fusion proteins and MYC were suppressed more profoundly upon combination treatment. Our findings provide a strong rationale for a novel targeted combination therapy that is expected to improve therapeutic outcomes in patients with MLL-rearranged leukemia.

The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

Mixed lineage leukemia (MLL) fusion-driven acute leukemias represent a genetically distinct subset of leukemias with poor prognosis. MLL forms a ternary complex with the lens epithelium-derived growth factor (LEDGF/p75) and MENIN. LEDGF/p75, a chromatin reader recognizing H3K36me3 marks, contributes to the association of the MLL multiprotein complex to chromatin. Formation of this complex is critical for the development of MLL leukemia. Available X-ray data represent only a partial structure of the LEDGF/p75-MLL-MENIN complex. Using nuclear magnetic resonance spectroscopy, we identified an additional LEDGF/ p75-MLL interface, which overlaps with the binding site of known LEDGF/p75 interactors-HIV-1 integrase, PogZ, and JPO2. Binding of these proteins or MLL to LEDGF/p75 is mutually exclusive. The resolved structure, as well as mutational analysis, shows that the interaction is primarily sustained via two aromatic residues of MLL (F148 and F151). Colony-forming assays in MLL-AF9 þ leukemic cells expressing MLL interaction-defective LEDGF/p75 mutants revealed that this interaction is essential for transformation. Finally, we show that the clonogenic growth of primary murine MLL-AF9-expressing leukemic blasts is selectively impaired upon overexpression of a LEDGF/p75-binding cyclic peptide CP65, originally developed to inhibit the LEDGF/p75-HIV-1 integrase interaction. The newly defined protein-protein interface therefore represents a new target for the development of therapeutics against LEDGF/p75-dependent MLL fusiondriven leukemic disorders. Cancer Res; 74(18); 5139-51. Ó2014 AACR.

BackgroundMedulloblastomas are malignant childhood brain tumors that arise due to the aberrant activity of developmental pathways during postnatal cerebellar development and in adult humans. Transcriptome analysis has identified four major medulloblastoma subgroups. One of them, the Sonic hedgehog (SHH) subgroup, is caused by aberrant Hedgehog signal transduction due to mutations in the Patched1 (PTCH1) receptor or downstream effectors. Mice carrying a Patched-1 null allele (Ptch1 ∆/+) are a good model to study the alterations underlying medulloblastoma development as a consequence of aberrant Hedgehog pathway activity.ResultsTranscriptome analysis of human medulloblastomas shows that SERPINE2, also called Protease Nexin-1 (PN-1) is overexpressed in most medulloblastomas, in particular in the SHH and WNT subgroups. As siRNA-mediated lowering of SERPINE2/PN-1 in human medulloblastoma DAOY cells reduces cell proliferation, we analyzed its potential involvement in medulloblastoma development using the Ptch1 ∆/+ mouse model. In Ptch1 ∆/+ mice, medulloblastomas arise as a consequence of aberrant Hedgehog pathway activity. Genetic reduction of Serpine2/Pn-1 interferes with medulloblastoma development in Ptch1 ∆/+ mice, as ~60% of the pre-neoplastic lesions (PNLs) fail to develop into medulloblastomas and remain as small cerebellar nodules. In particular the transcription factor Atoh1, whose expression is essential for development of SHH subgroup medulloblastomas is lost. Comparative molecular analysis reveals the distinct nature of the PNLs in young Ptch1 ∆/+ Pn-1 Δ/+ mice. The remaining wild-type Ptch1 allele escapes transcriptional silencing in most cases and the aberrant Hedgehog pathway activity is normalized. Furthermore, cell proliferation and the expression of the cell-cycle regulators Mycn and Cdk6 are significantly reduced in PNLs of Ptch1 ∆/+ Pn-1 Δ/+ mice.ConclusionsOur analysis provides genetic evidence that aberrant Serpine2/Pn-1 is required for proliferation of human and mouse medulloblastoma cells. In summary, our analysis shows that Serpine2/PN-1 boosts malignant progression of PNLs to medulloblastomas, in which the Hedgehog pathway is activated in a SHH ligand-independent manner.

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34 + hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1 −/− erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1 N1918Q SET-domain mutant induces terminal maturation of Nsd1 −/− erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSD N1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1 −/− erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.