Mammalian NDR kinases : tumor suppressors with essential functions in embryonic development

NDR kinases are highly conserved from yeast to man. Loss-of-function models of Ndr homologs in yeast and fly demonstrate essential functions of the respective kinases. Mammalian Ndr1 and Ndr2 are widely expressed and share a high degree of sequence identity. Human NDR kinases function in centriole duplication, mitotic chromosome alignment, apoptosis and proliferation. Mice that lack functional NDR1 protein are phenotypically normal, but protein levels of NDR2 are up-regulated in Ndr1-null tissues suggesting a compensatory link between both isoforms. Aged Ndr1 knock-out (KO) mice develop T-cell lymphoma, indicating a tumor suppressive function of mammalian NDR kinases. Several reports describe deregulated Ndr transcript levels in human cancers but the functional relevance of the expression changes has not been addressed. The present study reveals that mice carrying a targeted deletion of Ndr2 are phenotypically normal but show an up-regulation of NDR1 protein levels. Combined loss of Ndr1 and Ndr2 results in embryonic lethality, demonstrating that NDR kinases play essential roles in mammalian development. Ndr-null embryos are small and developmentally delayed at embryonic day (E) 8 and die around E10. Transcript levels of the CDK inhibitors p21 and p27 are up-regulated in Ndr-null embryos at E8.5, suggesting that NDR kinases positively regulate proliferation in vivo. Mutant somites are small and irregularly shaped. Asymmetric expression of the somite-clock genes Lunatic Fringe and Hes7 in mutant embryos indicates that NDR kinases contribute to ensure bilateral symmetry in the embryo. In the absence of NDR kinases, heart development arrests at the linear heart tube stage and does not proceed to cardiac looping. Proper establishment of the left / right axis is a prerequisite for rightward cardiac looping. Cardiac malformation is most likely the primary cause for embryonic lethality of Ndr-null embryos. Asymmetric gene expression and impaired cardiac looping might reflect a general symmetry defect in NDR-deficient embryos. Embryonic lethality precludes the analysis of in vivo functions of NDR kinases in the adult mouse. To address the role of NDR in the context of tumorigenesis, I have generated an intestinal epithelium specific Ndr1/2 double KO (Ndr1-/-Ndr2Δ/ΔVilCre) mouse line. Ndr1-/-Ndr2Δ/ΔVilCre mice develop rectal prolapse, a symptom of chronic inflammation of the colon. Importantly, patients suffering from chronic colitis are at increased risk of developing colorectal cancer (CRC). Although Ndr1-/-Ndr2Δ/ΔVilCre mice do not spontaneously develop colon cancer, initial studies indicate that Ndr1-/-Ndr2Δ/ΔVilCre mice are more susceptible to Azoxymethane (AOM)-induced colon carcinogenesis. Therefore, Ndr1-/-Ndr2Δ/ΔVilCre mice could provide a new model system to study the molecular mechanisms that underlie the increased risk of CRC formation in patients with chronic colonic inflammation. In summary, this study demonstrates that mammalian NDR kinases are essential for embryonic development. They positively regulate growth, somitogenesis and heart development. Whether the defect in bilateral symmetry and the cardiac phenotype are causally connected remains to be addressed. Complete loss of NDR kinases in the intestinal epithelium causes rectal prolapse and increased susceptibility to AOM-induced CRC formation. Lastly, the conditional Ndr double KO mouse line represents a valuable tool to address additional in vivo functions of mammalian NDR kinases in normal physiology and disease

<i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.

<p>Genotype (GT) distribution of offspring from <i>Ndr-</i>single allele intercrosses (<i>Ndr1</i>^+/-;<i>Ndr2</i>^-/- x <i>Ndr1</i>^-/-;<i>Ndr2</i>^+/-) at weaning. Actual offspring numbers are indicated, together with the expected and obtained Mendelian ratios. No <i>Ndr1/2</i>-double null mice were recovered. All other genotypes were obtained at approximately the expected Mendelian ratios. A total of 415 offspring were analyzed (n = 415).</p><p><i>Ndr1/2</i>-double null mice are embryonic lethal, but a single <i>Ndr</i> allele is sufficient to sustain normal development.</p

NDR kinases are essential for growth, cardiac development and blood vessel remodeling from about embryonic day 8 onward in mouse embryos.

<p>(A, B) Bright field images of wild-type (A) and <i>Ndr1/2</i>-double null (B) littermates at E8.5. Both embryos are of the 6-somite stage. Note that the <i>Ndr1/2</i>-double null somites are small and irregularly shaped. Scale bars correspond to 500μm. (C) Average somite numbers of wild-type and <i>Ndr1/2</i>-double null littermates at E8.5. Data correspond to the analysis of a total of 15 litters and are blotted as box and whisker chart illustrating the distribution of somite numbers per litter and genotype. Average somite numbers are indicated. (D, E) Distribution of <i>Shh</i> (D) and <i>T/brachyury</i> (E) transcripts in wild-type (left) and <i>Ndr1/2</i>-double null littermate embryos (right) at E8.5. Four animals per genotype were analyzed, and all embryos displayed the staining shown in Fig 3D and 3E. (F, G, H) Bright field images of wild-type (F) and <i>Ndr1/2</i>-double null (G, H) littermate embryos at E9.5. 56 <i>Ndr1/2</i>-double null and 163 control embryos at E9.5 were analyzed. White arrow in H points to the pericardial edema. Scale bars correspond to 500μm. (I, J) Bright field images of the yolk sacs of wild-type (I) and <i>Ndr1/2</i>-double null (J) littermate embryos at E9.5. All <i>Ndr1/2</i>-double null yolk sacs (n = 56) displayed defective vascular development as illustrated in Fig 3J.</p

Somitogenesis is altered in <i>Ndr1/2</i>-double null embryos.

<p>(A) Changes in gene expression between wild-type and <i>Ndr1/2</i>-double null embryos at E8.5 determined by microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s007" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s008" target="_blank">S2</a> Tables). (B, C, D) Distribution of <i>Meox1</i> (B), <i>Tbx6</i> (C) <i>and Mesogenin1</i> (D) transcripts in wild-type (wt) and <i>Ndr1/2</i>-double null embryos at E8.5. Four animals per genotype were analyzed for <i>Meox1</i> staining, and two embryos per genotype were analyzed for <i>Tbx6</i> and <i>Mesogenin</i> staining. All embryos displayed the staining patterns as shown in Fig 5B, 5C and 5D. (E) Hematoxylin/Eosin staining of transversal (left) and parasagittal (right) sections of wild-type (top panels) and <i>Ndr1/2</i>-double null (bottom panels) embryos at the 6-somite stage. Six embryos of each genotype were analyzed. PSM: presomitic mesoderm; SI, SII, SIII: last, second to last and third to last formed somite. Scale bars = 50μm. (F, G, H) Distribution of <i>Snail1</i> (F), <i>Axin2</i> (G) <i>and Lnfg</i> (H) transcripts in wild-type (wt) and <i>Ndr1/2</i>-double null embryos. Asterisk (*) indicates aberrant asymmetrical expression (right) of <i>Lnfg</i> in the last formed somite pair. Seven embryos were analyzed per genotype for <i>Snail1</i> staining, and five embryos were analyzed per genotype for <i>Axin2</i> staining. The expression patterns of <i>Snail1</i> and <i>Axin2</i> appeared indistinguishable between all mutant and control embryos. Nine mutant and four control embryos were analyzed for <i>Lnfg</i> staining. All four control embryos showed the expected symmetric expression pattern as illustrated in Fig 5H. In contrast, four of nine mutant embryos displayed strongly asymmetric <i>Lnfg</i> expression and one mutant embryo showed mild asymmetric expression.</p

<i>Ndr1/2</i>-double null embryos die around mid-gestation.

<p>Genotype distribution in embryos derived from intercrosses of <i>Ndr1</i>^+/-;<i>Ndr2</i>^-/- with <i>Ndr1</i>^-/-; <i>Ndr2</i>^+/- mice at indicated time points. Note (a): all <i>Ndr1/2</i>-double null embryos recovered at E10.5 were dead and were in progress of resorption.</p><p><i>Ndr1/2</i>-double null embryos die around mid-gestation.</p

Murine NDR kinases are essential for cardiac looping.

<p>(A) OPT 3D reconstruction of wild-type (Ai) and <i>Ndr1/2</i>-double null (Aii) embryos at E8.5. Blue: anatomy. Red: <i>Nkx2</i>.<i>5</i> whole mount <i>in situ</i> hybridization. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right. Two <i>Ndr1/2</i>-double null and two control embryos were examined. Using bright field microscopy ten <i>Ndr1/2</i>-double null and ten control embryos were analysed at E8.5 to confirm the observed phenotype (data not shown). (B, C) Bright field images of wild-type (Bi, Ci) and <i>Ndr1/2</i>-double null (Bii, Cii) developing hearts at E9.5. B: frontal view; C: lateral view. Scale bars = 100μm. Embryo axis orientation: a: anterior, p: posterior, l: left, r: right, d: dorsal, v: ventral. Five <i>Ndr1/2</i>-double null and five control embryos were analyzed. (D, E) OPT virtual section of wild-type (Di, Ei) and <i>Ndr1/2</i>-double null (Dii, Eii) embryos shown in 6A. Panel D: coronal plane; panel E: transversal plane. Labels: a: anterior, p: posterior, d: dorsal, v: ventral. Arrows point to the heart. Two <i>Ndr1/2</i>-double null and two control embryos were analyzed for OPT as shown in 6D and 6E. (F, G, H) Hematoxylin and Eosin stained transversal sections of a wild-type (Fi, Gi, Hi) and <i>Ndr1/2</i>-double null (Fi, Gi, Hi) hearts at the 6-somite stage. The myocardium (MC) and headfolds (HF) are indicated in (Fi) and (Fii). Arrows in (Fii), (Gii) and (Hii) point to remaining cells in the cardiac jelly and lumen. Note the similar section plan between the embryos shown in (E) and (G). Three <i>Ndr1/2</i>-double null and three control embryos were analyzed. (J) Scheme showing the approximate level of the sections within the embryo. The distance between sections is about 30μm.</p

The CDK inhibitor p21/Cip1 is up-regulated in <i>Ndr1/2</i>-double null embryos and required for survival.

<p>(A) Changes in transcript levels between wild-type and <i>Ndr1/2</i>-double null embryos at E8.5 as revealed by microarray analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s007" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136566#pone.0136566.s008" target="_blank">S2</a> Tables). (B) Validation of the alterations in <i>p21</i> and <i>p27</i> expression in wild-type (control) and <i>Ndr1/2</i>-double null embryos by qRT-PCR (at E8.5). Data shown represent the average gene expression levels obtained by analyzing three independent embryos per genotype. Each embryo was analyzed in triplicate. Statistical analysis was performed using a two-tailed Student t-test assuming unequal variance. (C) Genotype distribution of offspring from <i>Ndr1</i>^+/-;<i>Ndr2</i>^-/-;<i>p21</i>^+/- intercrosses. Embryos were isolated at E8.5 and genotyped. Top panel: <i>p21</i> genotype of the <i>Ndr1/2</i>-double null embryos; bottom panel: <i>Ndr1</i> genotype of the <i>p21</i>-null embryos. The p-values are indicative of the probabilities to obtain the observed genotype distribution by chance.</p

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration

Topical Bimiralisib Shows Meaningful Cutaneous Drug Levels in Healthy Volunteers and Mycosis Fungoides Patients but No Clinical Activity in a First-in-Human, Randomized Controlled Trial

Mycosis fungoides (MF) is a subtype of CTCL with a low incidence and high medical need for novel treatments. The objective of this randomized, placebo-controlled, double-blinded, first-in-human study was to evaluate safety, efficacy, cutaneous and systemic pharmacokinetics (PK) of topical bimiralisib in healthy volunteers (HVs) and MF patients. In this trial, a total of 6 HVs and 19 early-stage MF patients were treated with 2.0% bimiralisib gel and/or placebo. Drug efficacy was assessed by the Composite Assessment of Index Lesion Severity (CAILS) score, supported by objective measuring methods to quantify lesion severity. PK blood samples were collected frequently and cutaneous PK was investigated in skin punch biopsies on the last day of treatment. Local distribution of bimiralisib in HVs showed a mean exposure of 2.54 µg/g in the epidermis. A systemic concentration was observed after application of a target dose of 2 mg/cm2 on 400 cm2, with a mean Cavg of 0.96 ng/mL. Systemic exposure of bimiralisib was reached in all treated MF patients, and normalized plasma concentrations showed a 144% increased exposure compared to HVs, with an observed mean Cavg of 4.49 ng/mL and a mean cutaneous concentration of 5.3 µg/g. No difference in CAILS or objective lesion severity quantification upon 42 days of once-daily treatment was observed in the MF patient group. In general, the treatment was well tolerated in terms of local reactions as well as systemic adverse events. In conclusion, we showed that topical bimiralisib treatment leads to (i) meaningful cutaneous drug levels and (ii) well-tolerated systemic drug exposure in MF patients and (iii) a lack of clinical efficacy, in need of further exploration due to numerous unknown factors, before depreciation of topical bimiralisib as a novel therapeutic drug for CTCLs

Correction: Topical Bimiralisib Shows Meaningful Cutaneous Drug Levels in Healthy Volunteers and Mycosis Fungoides Patients but No Clinical Activity in a First-in-Human, Randomized Controlled Trial.(Cancers, (2022), 14, (1510), 10.3390/cancers14061510)

The authors wish to make the following corrections to this paper [1]: 1. ‘Harald Schnidar’ was not included as an author in the published article and he was added as the co-author including his affiliation 8. The corrected Author Contributions Statement was updated as follows: