Structural studies of intrinsically disordered MLL-fusion protein AF9 in complex with peptidomimetic inhibitors.

AF9 (MLLT3) and its paralog ENL(MLLT1) are members of the YEATS family of proteins with important role in transcriptional and epigenetic regulatory complexes. These proteins are two common MLL fusion partners in MLL-rearranged leukemias. The oncofusion proteins MLL-AF9/ENL recruit multiple binding partners, including the histone methyltransferase DOT1L, leading to aberrant transcriptional activation and enhancing the expression of a characteristic set of genes that drive leukemogenesis. The interaction between AF9 and DOT1L is mediated by an intrinsically disordered C-terminal ANC1 homology domain (AHD) in AF9, which undergoes folding upon binding of DOT1L and other partner proteins. We have recently reported peptidomimetics that disrupt the recruitment of DOT1L by AF9 and ENL, providing a proof-of-concept for targeting AHD and assessing its druggability. Intrinsically disordered proteins, such as AF9 AHD, are difficult to study and characterize experimentally on a structural level. In this study, we present a successful protein engineering strategy to facilitate structural investigation of the intrinsically disordered AF9 AHD domain in complex with peptidomimetic inhibitors by using maltose binding protein (MBP) as a crystallization chaperone connected with linkers of varying flexibility and length. The strategic incorporation of disulfide bonds provided diffraction-quality crystals of the two disulfide-bridged MBP-AF9 AHD fusion proteins in complex with the peptidomimetics. These successfully determined first series of 2.1-2.6 Å crystal complex structures provide high-resolution insights into the interactions between AHD and its inhibitors, shedding light on the role of AHD in recruiting various binding partner proteins. We show that the overall complex structures closely resemble the reported NMR structure of AF9 AHD/DOT1L with notable difference in the conformation of the ß-hairpin region, stabilized through conserved hydrogen bonds network. These first series of AF9 AHD/peptidomimetics complex structures are providing insights of the protein-inhibitor interactions and will facilitate further development of novel inhibitors targeting the AF9/ENL AHD domain.

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

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

In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets.

High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients' leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.

Discovery of a potent MLL1 and WDR5 protein-protein interaction inhibitor with in vivo antitumor activity.

MLL1-WDR5 interaction is essential for the formation of MLL core complex and its H3K4 methyltransferase activity. Disrupting MLL1-WDR5 interaction has been proposed as a potential therapeutic approach in the treatment of leukemia. A "toolkit" of well-characterized chemical probe will allow exploring animal studies. Based on a specific MLL1-WDR5 PPI inhibitor (DDO-2117), which was previously reported by our group, we conducted a bioisosterism approach by click chemistry to discover novel phenyltriazole scaffold MLL1-WDR5 interaction blockers. Here, our efforts resulted in the best inhibitor 24 (DDO-2093) with high binding affinity (Kd = 11.6 nM) and with improved drug-like properties. Both in vitro and in vivo assays revealed 24 could efficiently block the MLL1-WDR5 interaction. Furthermore, 24 significantly suppressed tumor growth in the MV4-11 xenograft mouse model and showed a favorable safety profile. We propose 24 as a chemical probe that is suitable for in vivo pharmacodynamic and biological studies of MLL1-WDR5 interaction.

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.

Menin­MLL inhibitors induce ferroptosis and enhance the anti­proliferative activity of auranofin in several types of cancer cells.

Menin­mixed­lineage leukemia (MLL) inhibitors have potential for use as therapeutic agents for MLL­rearranged leukemia. They are also effective against solid cancers, such as breast cancer. The present study demonstrated that menin­MLL inhibitors, such as MI­463, unexpectedly induced the ferroptotic cell death of several cancer cell lines. MI­463 at a double­digit nM concentration markedly decreased the viable number of OVCAR­8 ovarian cancer cells for 3 days. Ferrostatin­1 (a ferroptosis inhibitor) almost completely abrogated the MI­463­induced decrease in viable cell numbers. Furthermore, the cancer cell­killing activity was inhibited by N­acetylcysteine [a scavenger of reactive oxygen species (ROS)], deferoxamine (DFO, an iron chelator), PD146176 (a specific inhibitor of arachidonate 15­lipoxygenase), idebenone (a membrane­permeable analog of CoQ10) and oleic acid [a monounsaturated fatty acid and one of the end products of stearoyl­CoA desaturase 1 (SCD1)], whereas Z­VAD­FMK (an apoptosis inhibitor) had a negligible effect on cell death. It was also found that MI­463 in combination with auranofin (a thioredoxin reductase inhibitor) synergistically increased cancer the death of breast, ovarian, pancreatic and lung cancer cell lines (88%, 14/16 cell lines). The synergistic induction of cell death was abrogated by ferroptosis inhibitor and DFO. Inhibitors of SCD1, similar to MI­463, also enhanced cancer cell death synergistically with auranofin, while inhibitors of SCD1 and MI­463 did not additively induce cell death. Treatment with zinc protoporphyrin­9, a specific inhibitor of heme oxygenase­1 (HO­1), markedly attenuated the cell death induced by MI­463 plus auranofin. On the whole, these results suggest that the MI­463­induced decrease in cell viability may be at least partly associated with the inhibition of SCD1 activity. In addition, the potent induction of HO­1 contributed to the synergistic effects of MI­463 plus auranofin. Therefore, menin­MLL inhibitors, such as MI­463, in combination with auranofin represent an effective therapeutic approach for several types of cancer via the induction of ferroptosis.

Combined inhibition of menin-MLL interaction and TGF-ß signaling induces replication of human pancreatic beta cells.

Both type 1 and type 2 diabetes are associated with hyperglycemia and loss of functional beta cell mass. Inducing proliferation of preexisting beta cells is an approach to increase the numbers of beta cells. In this study, we examined a panel of selected small molecules for their proliferation-inducing effects on human pancreatic beta cells. Our results demonstrated that a small molecule inhibitor of the menin-MLL interaction (MI-2) and small molecule inhibitors of TGF-ß signaling (SB431542, LY2157299, or LY364947) synergistically increased ex vivo replication of human beta cells. We showed that this increased proliferation did not affect insulin production, as a pivotal indication of beta cell function. We further provided evidence which suggested that menin-MLL and TGF-ß inhibition cooperated through downregulation of cell cycle inhibitors CDKN1A, CDKN1B, and CDKN2C. Our findings might provide a new option for extending the pharmacological repertoire for induction of beta cell proliferation as a potential therapeutic approach for diabetes.

Synergistic targeting of FLT3 mutations in AML via combined menin-MLL and FLT3 inhibition.

The interaction of menin (MEN1) and MLL (MLL1, KMT2A) is a dependency and provides a potential opportunity for treatment of NPM1-mutant (NPM1mut) and MLL-rearranged (MLL-r) leukemias. Concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 occur in both leukemias and are particularly common in the NPM1mut subtype. In this study, transcriptional profiling after pharmacological inhibition of the menin-MLL complex revealed specific changes in gene expression, with downregulation of the MEIS1 transcription factor and its transcriptional target gene FLT3 being the most pronounced. Combining menin-MLL inhibition with specific small-molecule kinase inhibitors of FLT3 phosphorylation resulted in a significantly superior reduction of phosphorylated FLT3 and transcriptional suppression of genes downstream of FLT3 signaling. The drug combination induced synergistic inhibition of proliferation, as well as enhanced apoptosis, compared with single-drug treatment in models of human and murine NPM1mut and MLL-r leukemias harboring an FLT3 mutation. Primary acute myeloid leukemia (AML) cells harvested from patients with NPM1mutFLT3mut AML showed significantly better responses to combined menin and FLT3 inhibition than to single-drug or vehicle control treatment, whereas AML cells with wild-type NPM1, MLL, and FLT3 were not affected by either of the 2 drugs. In vivo treatment of leukemic animals with MLL-r FLT3mut leukemia reduced leukemia burden significantly and prolonged survival compared with results in the single-drug and vehicle control groups. Our data suggest that combined menin-MLL and FLT3 inhibition represents a novel and promising therapeutic strategy for patients with NPM1mut or MLL-r leukemia and concurrent FLT3 mutation.

Novel therapeutic strategies for MLL-rearranged leukemias.

MLL rearrangement is one of the key drivers and generally regarded as an independent poor prognostic marker in acute leukemias. The standard of care for MLL-rearranged (MLL-r) leukemias has remained largely unchanged for the past 50 years despite unsatisfying clinical outcomes, so there is an urgent need for novel therapeutic strategies. An increasing body of evidence demonstrates that a vast number of epigenetic regulators are directly or indirectly involved in MLL-r leukemia, and they are responsible for supporting the aberrant gene expression program mediated by MLL-fusions. Unlike genetic mutations, epigenetic modifications can be reversed by pharmacologic targeting of the responsible epigenetic regulators. This leads to significant interest in developing epigenetic therapies for MLL-r leukemia. Intriguingly, many of the epigenetic enzymes also involve in DNA damage response (DDR), which can be potential targets for synthetic lethality-induced therapies. In this review, we will summarize some of the recent advances in the development of epigenetic and DDR therapeutics by targeting epigenetic regulators or protein complexes that mediate MLL-r leukemia gene expression program and key players in DDR that safeguard essential genome integrity. The rationale and molecular mechanisms underpinning the therapeutic effects will also be discussed with a focus on how these treatments can disrupt MLL-fusion mediated transcriptional programs and impair DDR, which may help overcome treatment resistance.

Epigenetic regulation of protein translation in KMT2A-rearranged AML.

Inhibition of the H3K79 histone methyltransferase DOT1L has exhibited encouraging preclinical and early clinical activity in KMT2A (MLL)-rearranged leukemia, supporting the development of combinatorial therapies. Here, we investigated two novel combinations: dual inhibition of the histone methyltransferases DOT1L and EZH2, and the combination with a protein synthesis inhibitor. EZH2 is the catalytic subunit in the polycomb repressive complex 2 (PRC2), and inhibition of EZH2 has been reported to have preclinical activity in KMT2A-r leukemia. When combined with DOT1L inhibition, however, we observed both synergistic and antagonistic effects. Interestingly, antagonistic effects were not due to PRC2-mediated de-repression of HOXA9. HOXA cluster genes are key canonical targets of both KMT2A and the PRC2 complex. The independence of the HOXA cluster from PRC2 repression in KMT2A-r leukemia thus affords important insights into leukemia biology. Further studies revealed that EZH2 inhibition counteracted the effect of DOT1L inhibition on ribosomal gene expression. We thus identified a previously unrecognized role of DOT1L in regulating protein production. Decreased translation was one of the earliest effects measurable after DOT1L inhibition and specific to KMT2A-rearranged cell lines. H3K79me2 chromatin immunoprecipitation sequencing patterns over ribosomal genes were similar to those of the canonical KMT2A-fusion target genes in primary AML patient samples. The effects of DOT1L inhibition on ribosomal gene expression prompted us to evaluate the combination of EPZ5676 with a protein translation inhibitor. EPZ5676 was synergistic with the protein translation inhibitor homoharringtonine (omacetaxine), supporting further preclinical/clinical development of this combination. In summary, we discovered a novel epigenetic regulation of a metabolic process-protein synthesis-that plays a role in leukemogenesis and affords a combinatorial therapeutic opportunity.

Discovery of M-808 as a Highly Potent, Covalent, Small-Molecule Inhibitor of the Menin-MLL Interaction with Strong In Vivo Antitumor Activity.

Targeting the menin-MLL protein-protein interaction is a new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein the structure-based optimization of a class of covalent menin inhibitors, which led to the discovery of M-808 (16) as a highly potent and efficacious covalent menin inhibitor. M-808 effectively inhibits leukemia cell growth at low nanomolar concentrations and is capable of achieving partial tumor regression in an MV4;11 xenograft tumor model in mice at a well-tolerated dose schedule. Determination of the co-crystal structure of M-808 in complex with menin provides a structural basis for their high-affinity, covalent interactions. M-808 represents a promising, covalent menin inhibitor for further optimization and evaluation toward developing a new therapy for the treatment of MLL leukemia.

Proton pump inhibitors selectively suppress MLL rearranged leukemia cells via disrupting MLL1-WDR5 protein-protein interaction.

Genetic rearrangements of the mixed lineage leukemia (MLL) leading to oncogenic MLL-fusion proteins (MLL-FPs). MLL-FPs occur in about 10% of acute leukemias and are associated with dismal prognosis and treatment outcomes which emphasized the need for new therapeutic strategies. In present study, by a cell-based screening in-house compound collection, we disclosed that Rabeprazole specially inhibited the proliferation of leukemia cells harboring MLL-FPs with little toxicity to non-MLL cells. Mechanism study showed Rabeprazole down-regulated the transcription of MLL-FPs related Hox and Meis1 genes and effectively inhibited MLL1 H3K4 methyltransferase (HMT) activity in MV4-11 cells bearing MLL-AF4 fusion protein. Displacement of MLL1 probe from WDR5 protein suggested that Rabeprazole may inhibit MLL1 HMT activity through disturbing MLL1-WDR5 protein-protein interaction. Moreover, other proton pump inhibitors (PPIs) also indicated the inhibition activity of MLL1-WDR5. Preliminary SARs showed the structural characteristics of PPIs were also essential for the activities of MLL1-WDR5 inhibition. Our results indicated the drug reposition of PPIs for MLL-rearranged leukemias and provided new insight for further optimization of targeting MLL1 methyltransferase activity, the MLL1-WDR5 interaction or WDR5.

MLL1 Inhibition and Vitamin D Signaling Cooperate to Facilitate the Expanded Pluripotency State.

Dynamic establishment of histone modifications in early development coincides with programed cell fate restriction and loss of totipotency beyond the early blastocyst stage. Causal function of histone-modifying enzymes in this process remains to be defined. Here we show that inhibiting histone methyltransferase MLL1 reprograms naive embryonic stem cells (ESCs) to expanded pluripotent stem cells (EPSCs), with differentiation potential toward both embryonic and extraembryonic lineages in vitro and in vivo. MLL1 inhibition or deletion upregulates gene signatures of early blastomere development. The function of MLL1 in restricting induction of EPSCs is mediated partly by Gc, which regulates cellular response to vitamin D signaling. Combined treatment of MLL1 inhibitor and 1α,25-dihydroxyvitamin D3 (1,25-(OH)2D3) cooperatively enhanced functionality of EPSCs, triggering an extended 2C-like state in vitro and robust totipotent-like property in vivo. Our study sheds light on interplay between epigenetics and vitamin D pathway in cell fate determination.

Inhibition of the H3K4 methyltransferase MLL1/WDR5 complex attenuates renal senescence in ischemia reperfusion mice by reduction of p16INK4a.

Renal function declines with aging and is pathologically characterized by chronic inflammation and fibrosis. Renal senescence is induced not only by aging but also by various stimuli, including ischemia reperfusion injury. Recently, the accumulation of p16INK4a-positive cells in the kidney has been considered a molecular feature of renal senescence, with the p16INK4a gene reportedly regulated by mixed-lineage leukemia 1 (MLL1)/WD-40 repeat protein 5 (WDR5)-mediated histone 3 lysine 4 trimethylation (H3K4me3). Here, we determined whether inhibition of MLL1/WDR5 activity attenuates renal senescence, inflammation, and fibrosis in mice with ischemia reperfusion injury and in cultured rat renal fibroblasts. MM-102 or OICR-9429, both MLL1/WDR5 protein-protein interaction inhibitors, and small interfering RNA (siRNA) for MLL1 or WDR5 suppressed the expression of p16INK4a in mice with ischemia reperfusion injury, accompanied by downregulation of H3K4me3 expression. MM-102 attenuated renal fibrosis and inflammation in the kidney of mice with ischemia reperfusion injury. Moreover, in vitro study showed that transforming growth factor-ß1 induced the expression of MLL1, WDR5, H3K4me3, and p16INK4a. Finally, chromatin immunoprecipitation identified the p16INK4a promoter at an H3K4me3 site in renal fibroblasts. Thus, our findings show that H3K4me3 inhibition ameliorates ischemia reperfusion-induced renal senescence along with fibrosis and inflammation.

Genomic and Epigenomic Features of Primary and Recurrent Hepatocellular Carcinomas.

BACKGROUND & AIMS: Intratumor heterogeneity and divergent clonal lineages within and among primary and recurrent hepatocellular carcinomas (HCCs) produce challenges to patient management. We investigated genetic and epigenetic variations within liver tumors, among hepatic lesions, and between primary and relapsing tumors. METHODS: Tumor and matched nontumor liver specimens were collected from 113 patients who underwent partial hepatectomy for primary or recurrent HCC at 2 hospitals in Hong Kong. We performed whole-genome, whole-exome, or targeted capture sequencing analyses of 356 HCC specimens collected from multiple tumor regions and matched initial and recurrent tumors. We performed parallel DNA methylation profiling analyses of 95 specimens. Genomes and epigenomes of nontumor tissues that contained areas of cirrhosis or fibrosis were analyzed. We developed liver cancer cell lines that endogenously expressed a mutant form of TP53 (R249S) or overexpressed mutant forms of STAT3 (D170Y, K348E, and Y640F) or JAK1 (S703I and L910P) and tested the abilities of pharmacologic agents to reduce activity. Cells were analyzed by immunoblotting and chromatin immunoprecipitation with quantitative polymerase chain reaction. RESULTS: We determined the monoclonal origins of individual tumors using a single sample collection approach that captured more than 90% of mutations that are detected in all regions of tumors. Phylogenetic and phylo-epigenetic analyses revealed interactions and codependence between the genomic and epigenomic features of HCCs. Methylation analysis revealed a field effect in cirrhotic liver tissues that predisposes them to tumor development. Comparisons of genetic features revealed that 52% of recurrent HCCs derive from the clonal lineage of the initial tumor. The clonal origin if recurrent HCCs allowed construction of a temporal map of genetic alterations that associated with tumor recurrence. Activation of JAK signaling to STAT was a characteristic of HCC progression via mutations that associate with response to drug sensitivity. The combination of a mutation that increases the function of TP53 and the 17p chromosome deletion might provide liver cancer cells with a replicative advantage. Chromatin immunoprecipitation analysis of TP53 with the R249S substitution revealed its interaction with genes that encode chromatin regulators (MLL1 and MLL2). We validated MLL1 and MLL2 as direct targets of TP53R249S and affirmed their association in the Cancer Genome Atlas dataset. The MLL-complex antagonists MI-2-2 (inhibitor of protein interaction) and OICR-9492 (inhibitor of activity) specifically inhibited proliferation of HCC cells that express TP53R249S at nanomolar concentrations. CONCLUSIONS: We performed a systematic evaluation of intra- and intertumor genetic heterogeneity in HCC samples and identified genetic and epigenetic changes that associate with tumor progression and recurrence. We identified chromatin regulators that are upregulated by mutant TP53 in HCC cells and inhibitors that reduce proliferation of these cells. DNA methylation patterns in cirrhotic or fibrotic liver tissues might be used to identify those at risk of HCC development.

C-terminal BRE inhibits cellular proliferation and increases sensitivity to chemotherapeutic drugs of MLL-AF9 acute myeloid leukemia cells.

BRE (Brain and Reproductive Organ-Expressed) is an anti-apoptotic protein and a core component of DNA-repair BRCA1-A complex. Microarray-detected high BRE gene expression has been found to be associated with better patient survival in AML (acute myeloid leukemia) with MLL-AF9 translocation, and radiotherapy-treated non-familial breast cancer. A recent finding suggests that the high BRE gene expression in MLL-AF9 AML could be attributed to the additional expression of a transcript variant encoding a novel C-terminal BRE isoform. Using THP-1 as the MLL-AF9 AML cell model, we found that ectopic expression of the C-terminal BRE, which could not form an intact BRCA1-A complex, indeed increased cellular sensitivity to chemotherapeutic drugs and inhibited cell proliferation, while the complete opposite was achieved by the ectopic expression of full-length BRE. Our findings suggest that the C-terminal BRE-encoding transcript could be responsible for better patient survival and may have therapeutic potential for cancer.

CEBPA-mutated leukemia is sensitive to genetic and pharmacological targeting of the MLL1 complex.

The gene encoding the transcription factor C/EBPα is mutated in 10-15% of acute myeloid leukemia (AML) patients. N-terminal CEBPA mutations cause ablation of full-length C/EBPα without affecting the expression of a shorter oncogenic isoform, termed p30. The mechanistic basis of p30-induced leukemogenesis is incompletely understood. Here, we demonstrate that the MLL1 histone-methyltransferase complex represents a critical actionable vulnerability in CEBPA-mutated AML. Oncogenic C/EBPα p30 and MLL1 show global co-localization on chromatin and p30 exhibits robust physical interaction with the MLL1 complex. CRISPR/Cas9-mediated mutagenesis of MLL1 results in proliferation arrest and myeloid differentiation in C/EBPα p30-expressing cells. In line, CEBPA-mutated hematopoietic progenitor cells are hypersensitive to pharmacological targeting of the MLL1 complex. Inhibitor treatment impairs proliferation and restores myeloid differentiation potential in mouse and human AML cells with CEBPA mutations. Finally, we identify the transcription factor GATA2 as a direct critical target of the p30-MLL1 interaction. Altogether, we show that C/EBPα p30 requires the MLL1 complex to regulate oncogenic gene expression and that CEBPA-mutated AML is hypersensitive to perturbation of the MLL1 complex. These findings identify the MLL1 complex as a potential therapeutic target in AML with CEBPA mutations.

Impact of elevated anti-apoptotic MCL-1 and BCL-2 on the development and treatment of MLL-AF9 AML in mice.

Many acute myeloid leukaemias (AMLs) express high levels of BCL-2 and MCL-1, especially after therapy. To test the impact of these anti-apoptotic proteins on AML development and treatment, we used haemopoietic reconstitution to generate MLL-AF9 AMLs expressing BCL-2 or Mcl-1 transgenes. AMLs with elevated BCL-2 or MCL-1 had a higher proportion of mature myeloid cells but, like conventional MLL-AF9 AMLs, were readily transplantable. Short-term cell lines established from multiple primary AMLs of each genotype were tested in vitro for susceptibility to chemotherapeutics currently used for treating AML (daunorubicin, etoposide, cytarabine); the proteasome inhibitor bortezomib; CDK7/9 inhibitors; and BH3 mimetics, which bind and inhibit pro-survival proteins. The BH3 mimetics tested, alone and in combination with the other drugs, were: ABT-737 which, like its clinical counterpart navitoclax, targets BCL-2, BCL-XL and BCL-W; BCL-2-specific ABT-199 (venetoclax); BCL-XL-specific A-1331852; and S63845, a new MCL-1-specific BH3 mimetic. As single agents, daunorubicin and bortezomib had the greatest efficacy. Elevated MCL-1 or BCL-2 reduced sensitivity to daunorubicin but, surprisingly, not to bortezomib. MCL-1 markedly enhanced resistance to ABT-737 and ABT-199 but not S63845, and BCL-2 increased resistance to S63845 but not to ABT-737 or ABT-199. Notable synergies were achieved by combining BH3 mimetics with daunorubicin: S63845 increased the sensitivity of both MCL-1 and BCL-2 overexpressing MLL-AF9 AMLs, and ABT-737 aided in killing those overexpressing BCL-2. Synergy between daunorubicin and ABT-199 was also apparent in vivo, although not curative. Impressive synergistic responses were achieved for human MLL-fusion AML cell lines treated with daunorubicin plus either ABT-737, ABT-199 or S63845, and with ABT-199 plus S63845, with or without daunorubicin. Our data suggest that AML patients may benefit from combining conventional cytotoxic drugs with BH3 mimetics targeting BCL-2 or MCL-1 or, if tolerated, both these agents.

Combined Inhibition of DYRK1A, SMAD, and Trithorax Pathways Synergizes to Induce Robust Replication in Adult Human Beta Cells.

Small-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFßSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax- and SMAD-mediated transactivation of CDKN1C and CDKN1A. Notably, combined DYRK1A and TGFß inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.

Novel sub-cellular localizations and intra-molecular interactions may define new functions of Mixed Lineage Leukemia protein.

Mixed-lineage leukemia (MLL) protein is the best-characterized member of SET family of histone 3 lysine 4 methyltransferase, known for its transcriptional-activation role during development. mll gene rearrangements cause multiple kinds of aggressive leukemia in both children and adults. An important 'first' step in understanding the role of MLL in leukemogenesis would be to identify its localization throughout the cell cycle. In order to fully understand the breath of MLL functions in proliferating cells, we have analyzed its sub-cellular localization during the cell cycle. Our results show that MLL localizes to nucleolus and centrosome in interphase. During mitosis, it localizes to centrosomes and midbody in addition to previously reported spindle apparatus. Our results show that MLLN is required to translocate MLLC to the nucleolus. These finding suggest functional roles for MLL in nucleolus and mitosis. We also show how MLL-fusion proteins (MLL-FPs) localize to the same sub-cellular organelles like endogenous MLL. Our results indicate that MLL-fusion proteins may not only disturb the cell homeostasis by gain-of-function of the chimeric protein, but also by interfering with the functions of endogenous MLL.