Tfm Cells Research Articles

Lncrna Morrbid Regulates Myeloid Cell Survival of AML Driven By a Combination of FLT3ITD and Epigenetic Mutations

FLT3ITD mutations are observed in ~30% of all acute myeloid leukemia (AML) patients, primarily classified into M4/M5 subtype, and associated with poor survival. Frequently, FLT3ITD mutations are co-expressed with epigenetic mutations including TET2 and DNMT3A and portend a poor prognosis. We analyzed human RNA sequencing datasets for lncRNAs expressed in myeloid malignancies and identified novel lncRNAs including MORRBID. MORRBID is located close to BCL2L11, encoding the pro-apoptotic protein BIM. Human TCGA analysis showed MORRBID overexpression in AML patients with TET2 mutations and its association with poor overall survival. Our independent study using over 100 patients showed that AML patients with FLT3ITD mutation express significantly higher MORRBID compared to patients without such mutation. Consistently, mice lacking Tet2 or Dnmt3a but expressing Flt3ITD develop AML and express higher levels of Morrbid in Lin- Sca-1+ Kit+ (LSK) cells. We hypothesized that MORRBID may not only act as a novel prognostic and diagnostic biomarker for AML but also as a therapeutic target in AML bearing a combination of Tet2-/-;FLT3ITD/ITD (TF) and Dnmt3a-/-;FLT3ITD/ITD (DF) mutations. We generated Tet2-/-;Flt3ITD/ITD;Morrbid-/-(TFM)and Dnmt3a-/-;FLT3ITD/ITD;Morrbid-/-(DFM)mice and assessed their hematologic parameters. Loss of Morrbid in the context of TF and DF rescued majority of peripheral blood (PB) abnormalities associated with these models of AML including increased myeloid cells (neutrophils and monocytes). In addition, the infiltration of leukemia cells in lung was significantly removed in the setting of Morrbid loss. Mechanistically, a significant reduction in the expression of Bim was observed in Lin- cells derived from TF and DF mice, which was associated with increased expression of Morrbid. However, Morrbid loss increased expression of Bim, induced apoptosis and reversed the enhanced survival seen in LSK cells derived from TF and DF AML mice. Overall, the increased bone marrow (BM) cellularity observed in TF primary mice was normalized to WT levels in the absence of Morrbid, which was associated with an increase in apoptosis in both BM myeloid progenitors as well as in more mature myeloid cells. To determine if in a competitive setting, loss of Morrbid in the setting of TF leukemia stem cells would alter clonal hematopoiesis, overall engraftment and AML development, we performed a competitive transplant assay and observed complete correction in term of abnormal leukemia cell counts, including correction in PB counts, presence of Mac-1+ blasts in PB as well as overall engraftment of TFM cells 24 weeks post-transplant. The rescue in these defects associated with TF donor cells in the absence of Morrbid was observed in the PB, BM and spleen. Loss of Morrbid mitigates the clonal expansion of AML donor cells in both primary and secondary competitive transplant assays. Transplantation of whole BM cells bearing TFM cells resulted in a partial correction of the AML phenotype including correction of mature myeloid cells in PB and BM of the recipients. Importantly, the absolute number of leukemia progenitor cells, which are normally increased in the BM with TF donors, were also restored to WT levels in recipient mice with TFM donors. Finally, the reduced overall survival defects seen upon secondary transplantation of TF cells was significantly prolonged in the absence of Morrbid. Our results warrant that MORRBID is a novel prognostic/diagnostic biomarker and a therapeutic target for AML. Disclosures Carroll: Janssen Pharmaceuticals: Consultancy; Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding.

N-Cadherin expression in motoneurons is directly regulated by androgens: a genetic mosaic analysis in rats

We have recently reported that systemic androgens regulate adult N-cadherin (N-cad) expression in spinal motoneurons. However, the mechanism through which androgen mediates this effect remains undetermined. Androgen may act directly on motoneurons to regulate N-cad expression, or indirectly, via effects on androgen-sensitive afferent or efferent structures. Here, we describe a genetic mosaic investigation of this site-of-action indeterminacy. Following developmental random X chromosome inactivation, androgenized female rats heterozygous for the tfm androgen receptor mutation (X WTX tfm ) are phenotypic mosaics of androgen-sensitive wild-type (WT) and androgen-insensitive ( tfm) motoneurons. We compared steroid effects on WT and tfm cells in two sexually-dimorphic motoneuron pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), as well as a less steroid responsive motoneuron pool, the sexually monomorphic retrodorsolateral nucleus (RDLN). Independent of steroid treatment, a greater proportion of wild-type cells were N-cad immunoreactive (IR) in the DLN and RDLN. Following testosterone treatment, increased N-cad expression was observed in both cell types in the DLN, but in the SNB only the androgen-competent WT cells increased N-cad expression. Testosterone treatment did not significantly alter N-cad expression in the mosaic RDLN. The results indicate both cell autonomous and cell non-autonomous androgenic regulation of N-cad expression in spinal motoneurons.

Direct and mediated effects of testosterone: analysis of sex reversed mosaic mice heterozygous for testicular feminization

Sex reversed mice are XX males carrying on one of their X chromosomes a translocation of the sex determining region of the Y (Cattanach’s Sxr factor). The phenotype corresponds to the Klinefelter syndrome in man. The X linked Tfm (testicular feminization) mutation in the mouse is a frame shift in the androgen receptor gene leading to complete androgen insensitivity. Due to random X inactivation, sex reversed mice heterozygous for Tfm, are mosaics composed of a variable proportion of androgen insensitive XTfm and androgen sensitive X⁺ wildtype cells. In the intersexual genital tract, Tfm cells are maintained as undifferentiated cells in the epididymal duct. To the distorted prostate lobes and bulbourethral glands they contribute some lobules of indifferent urethral glands. A large contribution of Tfm cells allows downgrowth of Wolffian and Müllerian ducts to form a vagina. In the external genitalia the stimulatory effect of testosterone is reduced leading to various degrees of feminization correlating with the proportion of Tfm cells present. In the mosaics effects of testosterone, mediated by local growth factors from the wildtype to the Tfm cells, can be distinguished from direct effects expressed only in the wildtype cells. Mediated effects are embryonic induction and morphogenesis of male organs and postnatal maintenance of organ structure and proliferation. The direct effect is cellular differentiation.

Growth hormone secretion by individual somatotropes of the testicular feminized rat.

To investigate the cellular mechanisms underlying the unique GH secretory apparatus of the androgen-resistant testicular feminized (Tfm) rat we employed a reverse hemolytic plaque assay to assess GH secretion by individual cells from normal male, normal female, and Tfm rats. Acutely dispersed pituitary cells were incubated for 90 min with GH anti-serum in the presence of medium alone, 0.01, 0.1, 1, 10, or 100 nM GHRH, or 3 microM forskolin after which hemolytic plaques were developed over an additional 30 min. Body weights of the Tfm rats [318 +/- 7 g (mean +/- SEM)] were intermediate between intact males (372 +/- 18 g) and females (218 +/- 7 g). The total number of cells recovered from dispersion of Tfm rat pituitaries [3.20 +/- 0.42 X 10(6) (mean +/- SEM)] was greater than that from males (1.43 +/- 0.12 X 10(6); P = 0.001), but not distinguishable from that from females (2.31 +/- 0.30 X 10(6); P = 0.06). However, the absolute population of recovered somatotropes from the Tfm animals (1.24 +/- 0.22 X 10(6) exceeded both male (0.56 +/- 0.10 X 10(6); P = 0.002) and female (0.80 +/- 0.14 X 10(6); P = 0.046) values. Mean basal and maximal GH plaque areas were greater for cells from male rats than for those from either female or Tfm rats (P less than 0.05) regardless of whether GHRH or forskolin was used as the secretagogue. Plaque areas from female and Tfm cells were indistinguishable under all study conditions. These data suggest that a deficiency of androgen receptors prevents establishment of the greater GH secretory capacity of individual somatotropes characteristic of the adult male rat. This androgen receptor-dependent modulation of GH secretory capacity appears to occur at a step distal to the GHRH receptor. The data also suggest that an increase in the absolute population of somatotropes is an additional consequence of androgen receptor deficiency. This combination of individual somatotropes, each possessing a GH secretory capacity similar to that of cells from normal females, but present in greater absolute numbers, may explain the intermediate values found during previous studies of the Tfm rat GH axis which were based on assessment of large mixed populations of pituitary cells.

Intersex mice composed of androgen insensitive Tfm and wild-type cells analysed by 3H dihydrotestosterone autoradiography.

The X-linked testicular feminization mutation (Tfm) in the mouse is characterized by an androgen receptor defect. Due to random X-chromosome inactivation, XTfm/X+ heterozygotes are mosaics with respect to Tfm. They are composed of androgen receptor deficient XTfm cells and normal X+ wild-type cells. If Tfm heterozygotes are converted to XX males by the sex reversal factor (Sxr) the mosaicism is expressed. Therefore in sex reversed Tfm heterozygotes (XTfm/X+-Sxr) intersexual sex organs develop. In five intersexes with small male accessory glands and hypospadia and one heavily feminized intersex with vagina and caudally dislocated deferent ducts the mosaic is visualized by 3H-DHT-autoradiography. In the epididymis differentiated wild-type cells show nuclear labeling, whereas undifferentiated Tfm cells are unlabeled. Unlabeled Tfm cells are also encountered in the vesicular glands of the heavily feminized animal, demonstrating that Tfm cells can participate in the formation of male sex glands. The urethral glands of the mosaic animals are composed of unlabeled Tfm lobules exhibiting the female phenotype of the glands, and of labeled wild-type lobules exhibiting the male phenotype. Formation of a vagina and deviation of the deferent ducts is correlated with lack of androgen binding sites in the connective tissue.

Androgen receptor-deficient Tfm cells in the mosaic epididymis of sex-reversed mice heterozygous for Tfm: an autoradiographic study with [3H]-dihydrotestosterone and [3H]-estradiol.

Testicular feminization (Tfm) in the mouse is characterized by androgen insensitivity of the target cells. We describe the presence of androgen-insensitive Tfm cells in the epididymis of mosaic mice produced by converting female carriers of the Tfm mutation (XTfm/X+) to males via the sex reversal factor (Sxr). The mosaic arises by random X-inactivation. In the epididymal duct, flat undifferentiated Tfm cells are interspersed between high columnar wild-type cells. By thaw-mount autoradiography we show that after injection of [3H]dihydrotestosterone, radioactivity is concentrated in the nuclei of high columnar wild-type cells, whereas the nuclei of the low cuboid Tfm cells remain free of nuclear radioactivity. After injection of [3H]estradiol, both Tfm and wild-type cells show nuclear labeling. Our observations demonstrate that Tfm cells in the mosaic epididymis selectively lack nuclear dihydrotestosterone-binding sites, whereas estradiol-binding sites are intact.

Sex determination and sex differentiation

Sex determination in mammals proceeds like a cascade from the level of the sex chromosomes to the gonads, to the genital ducts, and finally to the expression of the male or female phenotype. At the level of the genital ducts male sex organs are induced by testosterone. Its action depends on an intact cytoplasmic androgen receptor protein. The testicular feminization mutation (tfm) leads to loss of hormone binding capacity. Individuals with testes but female external phenotype develop. In the mouse the interaction of androgen insensitive Tfm cells with normal cells can be studied in mosaic individuals composed of both cell types, and in organ culture by recombination of Tfm and normal tissues. The experiments show that under the action of testosterone the normal cells express male differentiated cellular functions, whereas the Tfm cells differentiate in female direction. In respect to proliferation and expression of male or female organ structures, however, both cell types communicate via local factors. Thus, instead of irregular malformations intersex organs develop.

Intact testosterone receptor complex does not induce RNA synthesis of Tfm-nuclei in multinucleated urethral muscle fibres of mosaic mice.

The X-linked testicular feminization mutation (Tfm) of the mouse leads to androgen insensitivity of target cells. Through the autosomal sex reversed (Sxr) factor we have converted female heterozygotes into males. Due to X-inactivation, mosaic animals arise which are composed of androgen sensitive wild-type and androgen insensitive Tfm cells. In the androgen dependent striated urethral muscle, Tfm and wild-type cells fuse and form multinucleated muscle fibres. In the muscle fibres the Tfm nuclei are exposed to the intact cytoplasmic testosterone receptor complex coded for by the wild-type nuclei. We ask the question whether under these conditions RNA synthesis can be stimulated in the Tfm nuclei. Castrated mosaic animals were injected with testosterone, and incorporation of 3H-uridine was studied by autoradiography. We found two classes of muscle cell nuclei, those with low grain counts corresponding to the Tfm controls and those with high grain counts corresponding to the stimulated male controls. The results indicate that the Tfm nuclei are not stimulated by the intact testosterone receptor complex.

Cell death in the mosaic epididymis of sex reversed mice, heterozygous for testicular feminization.

Sex reversed mice heterozygous for testicular feminization are genetic females which are converted to males by the autosomal Sxr ("sex reversed") mutation, thus imitating the Y-chromosome. They carry on one of their X-chromosomes the mutation for testicular feminization. Then, X-inactivation leads to the formation of a mosaic of androgen insensitive XTfm and androgen sensitive X+ cells. In the epididymis the Tfm cells are maintained into adulthood as undifferentiated flat cells. There are however, always less Tfm cells than expected from random X-inactivation. Therefore, in the present study the role of cell death in the modification of the original mosaic is investigated before and after castration. It is shown that survival or elimination of Tfm cells depends on the structure of the smooth muscle layers around the epididymal duct. In addition to elimination of whole Tfm sections, a steady loss of single cells, not only in the Tfm but also in the wild-type fraction occurs. In the connective tissue distinct necrotic areas are present. After castration massive necroses appear in both cell types. In Tfm sections disintegration of the wall layers again leads to elimination of Tfm cells and to protrusion of epithelial cells in the interstitium which later on may be organized as abnormal sprouts and ducts. The observations indicate that the testosterone dependent trophic principle is based on short-range intercellular interactions and presumably arises in the connective tissue component of the epididymis.

Cell interactions in the sex organs of sex reversed mice heterozygous for testicular feminization.

The X-linked mutation for testicular feminization (Tfm) described by Lyon and Hawkes (1970) causes androgen insensitivity of the testosterone target cells. The molecular basis of the phenotype is a defect of the androgen receptor protein (Attardi and Ohno, 1974; Gehring and Tomkins, 1974). Due to random X inactivation, XX mice heterozygous for Tfm are mosaics with respect to androgen sensitivity. They can be converted to males by the autosomal dominant mutation for “sex reversal” (Sxr) described by Cattanach, Pollard, and Hawkes (1971). By imitating the Y-chromosome, Sxr induces formation of testes in XX embryos. The testosterone produced by the embryonic testes in turn induces formation of male sex organs. In sex-reversed embryos heterozygous for Tfm, testosterone encounters a mixed population of androgen-insensitive and androgen-sensitive cells. In the androgen-insensitive cells the X chromosome bearing Tfm is active. The Tfm cells express the defective receptor protein and therefore are constitutively bound to form female organs. In the androgen-sensitive cells the wild-type X is active. The wild-type cells therefore express an intact androgen receptor protein. They respond to testosterone and are bound to form male sex organs.

Metabolic cooperation between Tfm and wild-type cells in mosaic mice after induction of DNA synthesis

Mosaic mice composed of androgen-insensitive Tfm and androgen-sensitive wild-type cells are constructed by virtue of the natural X inactivation: XX mice heterozygous for X-linked testicular feminization ( Tfm) are reverted to males by the sex reversal ( Sxr) mutation. After stimulation with testosterone, in the epididymis of the mosaic mice, the incorporation of 3H-thymidine is compared in both cell fractions. The labeling index of Tfm and wild-type cells is in the same order of magnitude. The result indicates that the stimulus for DNA synthesis exerted by testosterone is conveyed from the androgen-sensitive wild-type to the androgen-insensitive Tfm cells by metabolic cooperation on tissue level. On the other hand, the proportion of Tfm cells in the epididymal mosaic is far less than expected from X inactivation. The reason is that in the mosaic, in spite of normal proliferation, the undifferentiated Tfm cells die off steadily. Typical dense bodies are observed as signs of physiological cell death.

X-inactivation pattern in the epididymis of sex-reversed mice heterozygous for testicular feminization

ABSTRACT Female mice heterozygous for testicular feminization were sex-reversed by means of the autosomal sex reversal mutation (Sxr). Due to X-inactivation, the blastemata for male sex organs in these animals are composed of a mixture of cells, carrying either the wildtype X chromosome or the X chromosome affected with Tfm in an active state. Thus, the two types of cells are sensitive to androgens or insensitive to androgens, respectively. This mosaic could be demonstrated in the epididymis on a cellular level. Segments of undifferentiated Tfm cells were found alternating with normally differentiated wild-type cells. The ultras truc tura] appearance of the mosaic is described.

Genetically directed preferential X-activation seen in mice

The nature of the O hv mutation of the X chromosome of the mouse is defined. This locus exerts a cis position effect upon the expression of genes Tfm and Blo mapping in the same region; genes on the same chromosome as the O hv mutation are preferentially activated and genes on the other X chromosome are usually inactive. Following the proportion of Tfm cells in the kidneys of heterozygotes confirms that the variegation seen of the locus Blo in the coat is matched in inner tissues. By introducing the sex reversal gene Sxr into these stocks, a situation can be created in which wildtype kidney cells have a selective advantage over Tfm cells in the embryonic Wolfflan duct and urogenital sinus. In spite of this difference in cell advantages, Blo coat variegation and Tfm Wolffian duct cell preponderance continue to exhibit a good correlation. This excludes the possibility that the variegation depends upon a selective advantage of cells carrying Tfm alleles after random X-inactivation and therefore reinforces the conclusion that the O hv mutation is directly concerned in the X-activation process. A model is presented in which this locus acts as a receptor site recognized by molecules which activate one X chromosome.