Molecular synergy underlies the co-occurrence patterns and phenotype of NPM1-mutant acute myeloid leukemia.

NPM1 mutations define the commonest subgroup of acute myeloid leukemia (AML) and frequently co-occur with FLT3 internal tandem duplications (ITD) or, less commonly, NRAS or KRAS mutations. Co-occurrence of mutant NPM1 with FLT3-ITD carries a significantly worse prognosis than NPM1-RAS combinations. To understand the molecular basis of these observations, we compare the effects of the 2 combinations on hematopoiesis and leukemogenesis in knock-in mice. Early effects of these mutations on hematopoiesis show that compound Npm1cA/+;NrasG12D/+ or Npm1cA;Flt3ITD share a number of features: Hox gene overexpression, enhanced self-renewal, expansion of hematopoietic progenitors, and myeloid differentiation bias. However, Npm1cA;Flt3ITD mutants displayed significantly higher peripheral leukocyte counts, early depletion of common lymphoid progenitors, and a monocytic bias in comparison with the granulocytic bias in Npm1cA/+;NrasG12D/+ mutants. Underlying this was a striking molecular synergy manifested as a dramatically altered gene expression profile in Npm1cA;Flt3ITD , but not Npm1cA/+;NrasG12D/+ , progenitors compared with wild-type. Both double-mutant models developed high-penetrance AML, although latency was significantly longer with Npm1cA/+;NrasG12D/+ During AML evolution, both models acquired additional copies of the mutant Flt3 or Nras alleles, but only Npm1cA/+;NrasG12D/+ mice showed acquisition of other human AML mutations, including IDH1 R132Q. We also find, using primary Cas9-expressing AMLs, that Hoxa genes and selected interactors or downstream targets are required for survival of both types of double-mutant AML. Our results show that molecular complementarity underlies the higher frequency and significantly worse prognosis associated with NPM1c/FLT3-ITD vs NPM1/NRAS-G12D-mutant AML and functionally confirm the role of HOXA genes in NPM1c-driven AML.

Accelerated Development With Increased Bone Mass and Skeletal Response to Loading Suggest Receptor Activity Modifying Protein-3 as a Bone Anabolic Target.

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression ß-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically.

SETBP1 overexpression acts in the place of class-defining mutations to drive FLT3-ITD-mutant AML.

Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD-positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.

Research resource: Haploinsufficiency of receptor activity-modifying protein-2 (RAMP2) causes reduced fertility, hyperprolactinemia, skeletal abnormalities, and endocrine dysfunction in mice.

Receptor activity-modifying protein-2 (RAMP2) is a single-pass transmembrane protein that can regulate the trafficking, ligand binding, and signaling of several G protein-coupled receptors (GPCR). The most well-characterized role of RAMP2 is in the regulation of adrenomedullin (AM) binding to calcitonin receptor-like receptor (CLR), and our previous studies using knockout mouse models support this canonical signaling paradigm. For example, Ramp2(-/-) mice die at midgestation with a precise phenocopy of the AM(-/-) and Calcrl(-/-) mice. In contrast, Ramp2(+/-) mice are viable and exhibit an expanded variety of phenotypes that are distinct from those of Calcrl(+/-) mice. Using Ramp2(+/-) female mice, we demonstrate that a modest decrease in Ramp2 expression causes severe reproductive defects characterized by fetal growth restriction, fetal demise, and postnatal lethality that is independent of the genotype and gender of the offspring. Ramp2(+/-) female mice also exhibit hyperprolactinemia during pregnancy and in basal conditions. Consistent with hyperprolactinemia, Ramp2(+/-) female mice have enlarged pituitary glands, accelerated mammary gland development, and skeletal abnormalities including delayed bone development and decreased bone mineral density. Because RAMP2 has been shown to associate with numerous GPCR, it is likely that signaling of one or more of these GPCR is compromised in Ramp2(+/-) mice, yet the precise identification of these receptors remains to be elucidated. Taken together, this work reveals an essential role for RAMP2 in endocrine physiology and provides the first in vivo evidence for a physiological role of RAMP2 beyond that of AM/CLR signaling.