Menin inhibitor MI-3454 induces remission in MLL1-rearranged and NPM1-mutated models of leukemia.

The protein-protein interaction between menin and mixed lineage leukemia 1 (MLL1) plays a critical role in acute leukemias with translocations of the MLL1 gene or with mutations in the nucleophosmin 1 (NPM1) gene. As a step toward clinical translation of menin-MLL1 inhibitors, we report development of MI-3454, a highly potent and orally bioavailable inhibitor of the menin-MLL1 interaction. MI-3454 profoundly inhibited proliferation and induced differentiation in acute leukemia cells and primary patient samples with MLL1 translocations or NPM1 mutations. When applied as a single agent, MI-3454 induced complete remission or regression of leukemia in mouse models of MLL1-rearranged or NPM1-mutated leukemia, including patient-derived xenograft models, through downregulation of key genes involved in leukemogenesis. We also identified MEIS1 as a potential pharmacodynamic biomarker of treatment response with MI-3454 in leukemia, and demonstrated that this compound is well tolerated and did not impair normal hematopoiesis in mice. Overall, this study demonstrates, for the first time to our knowledge, profound activity of the menin-MLL1 inhibitor as a single agent in clinically relevant PDX models of leukemia. These data provide a strong rationale for clinical translation of MI-3454 or its analogs for leukemia patients with MLL1 rearrangements or NPM1 mutations.

MIR142 Loss-of-Function Mutations Derepress ASH1L to Increase HOXA Gene Expression and Promote Leukemogenesis.

Point mutations in the seed sequence of miR-142-3p are present in a subset of acute myelogenous leukemia (AML) and in several subtypes of B-cell lymphoma. Here, we show that mutations associated with AML result both in loss of miR-142-3p function and in decreased miR-142-5p expression. Mir142 loss altered the hematopoietic differentiation of multipotent hematopoietic progenitors, enhancing their myeloid potential while suppressing their lymphoid potential. During hematopoietic maturation, loss of Mir142 increased ASH1L protein expression and consequently resulted in the aberrant maintenance of Hoxa gene expression in myeloid-committed hematopoietic progenitors. Mir142 loss also enhanced the disease-initiating activity of IDH2-mutant hematopoietic cells in mice. Together these data suggest a novel model in which miR-142, through repression of ASH1L activity, plays a key role in suppressing HOXA9/A10 expression during normal myeloid differentiation. AML-associated loss-of-function mutations of MIR142 disrupt this negative signaling pathway, resulting in sustained HOXA9/A10 expression in myeloid progenitors/myeloblasts and ultimately contributing to leukemic transformation.Significance: These findings provide mechanistic insights into the role of miRNAs in leukemogenesis and hematopoietic stem cell function. Cancer Res; 78(13); 3510-21. ©2018 AACR.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Mammalian Kinesin-3 motors are dimeric in vivo and move by processive motility upon release of autoinhibition.

Kinesin-3 motors drive the transport of synaptic vesicles and other membrane-bound organelles in neuronal cells. In the absence of cargo, kinesin motors are kept inactive to prevent motility and ATP hydrolysis. Current models state that the Kinesin-3 motor KIF1A is monomeric in the inactive state and that activation results from concentration-driven dimerization on the cargo membrane. To test this model, we have examined the activity and dimerization state of KIF1A. Unexpectedly, we found that both native and expressed proteins are dimeric in the inactive state. Thus, KIF1A motors are not activated by cargo-induced dimerization. Rather, we show that KIF1A motors are autoinhibited by two distinct inhibitory mechanisms, suggesting a simple model for activation of dimeric KIF1A motors by cargo binding. Successive truncations result in monomeric and dimeric motors that can undergo one-dimensional diffusion along the microtubule lattice. However, only dimeric motors undergo ATP-dependent processive motility. Thus, KIF1A may be uniquely suited to use both diffuse and processive motility to drive long-distance transport in neuronal cells.

Fascin, a novel target of beta-catenin-TCF signaling, is expressed at the invasive front of human colon cancer.

Cancer cells become metastatic by acquiring a motile and invasive phenotype. This step requires remodeling of the actin cytoskeleton and the expression of exploratory, sensory organelles known as filopodia. Aberrant beta-catenin-TCF target gene activation plays a major role in colorectal cancer development. We identified fascin1, a key component of filopodia, as a target of beta-catenin-TCF signaling in colorectal cancer cells. Fascin1 mRNA and protein expression were increased in primary cancers in a stage-dependent manner. Fascin1 was exclusively localized at the invasive front of tumors also displaying nuclear beta-catenin. Forced expression of fascin1 in colorectal cancer cells increased their migration and invasion in cell cultures and caused cell dissemination and metastasis in vivo, whereas suppression of fascin1 expression by small interfering RNA reduces cell invasion. Although expression of fascin1 in primary tumors correlated with the presence of metastases, fascin1 was not expressed in metastases. Our studies show that fascin1 expression is tightly regulated during development of colon cancer metastases and is a novel target of beta-catenin-TCF signaling. We propose that transient up-regulation of fascin1 in colorectal cancer promotes the acquisition of migratory and invasive phenotypes that lead to metastasis. Moreover, the expression of fascin1 is down-regulated when tumor cells reach their metastatic destination where migration ceases and proliferation is enhanced. Although metastasis to vital organs is often the cause of mortality, only limited success has been attained in developing effective therapeutics against metastatic disease. We propose that genes involved in cell migration and invasion, such as fascin1, could serve as novel targets for metastasis prevention.

Two binding partners cooperate to activate the molecular motor Kinesin-1.

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.

Microtubule acetylation promotes kinesin-1 binding and transport.

Long-distance intracellular delivery is driven by kinesin and dynein motor proteins that ferry cargoes along microtubule tracks . Current models postulate that directional trafficking is governed by known biophysical properties of these motors-kinesins generally move to the plus ends of microtubules in the cell periphery, whereas cytoplasmic dynein moves to the minus ends in the cell center. However, these models are insufficient to explain how polarized protein trafficking to subcellular domains is accomplished. We show that the kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) is localized to only a subset of neurites in cultured neuronal cells. The mechanism of polarized trafficking appears to involve the preferential recognition of microtubules containing specific posttranslational modifications (PTMs) by the kinesin-1 motor domain. Using a genetic approach to eliminate specific PTMs, we show that the loss of a single modification, alpha-tubulin acetylation at Lys-40, influences the binding and motility of kinesin-1 in vitro. In addition, pharmacological treatments that increase microtubule acetylation cause a redirection of kinesin-1 transport of JIP1 to nearly all neurite tips in vivo. These results suggest that microtubule PTMs are important markers of distinct microtubule populations and that they act to control motor-protein trafficking.