Multiple myeloma: Combination therapy of BET proteolysis targeting chimeric molecule with CDK9 inhibitor.

Cyclin Dependent Kinase 9 (CDK9) associates with Bromodomain and Extra-Terminal Domain (BET) proteins to promote transcriptional elongation by phosphorylation of serine 2 of RNAP II C-terminal domain. We examined the therapeutic potential of selective CDK9 inhibitors (AZD 4573 and MC180295) against human multiple myeloma cells in vitro. Short-hairpin RNA silencing of CDK9 in Multiple Myeloma (MM) cell lines reduced cell viability compared to control cells showing the dependency of MM cells on CDK9. In order to explore synergy with the CDK9 inhibitor, proteolysis targeting chimeric molecule (PROTAC) ARV 825 was added. This latter drug causes ubiquitination of BET proteins resulting in their rapid and efficient degradation. Combination treatment of MM cells with ARV 825 and AZD 4573 markedly reduced their protein expression of BRD 2, BRD 4, MYC and phosphorylated RNA pol II as compared to each single agent alone. Combination treatment synergistically inhibited multiple myeloma cells both in vitro and in vivo with insignificant weight loss. The combination also resulted in marked increase of apoptotic cells at low dose compared to single agent alone. Taken together, our studies show for the first time that the combination of a BET PROTAC (ARV 825) plus AZD 4573 (CDK9 inhibitor) is effective against MM cells.

Proteolysis targeting chimeric molecules as therapy for multiple myeloma: efficacy, biomarker and drug combinations.

Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients.

Sonographic Alteration of Basal Ganglia in Different Forms of Primary Focal Dystonia: A Cross-sectional Study / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Few studies have addressed whether abnormalities in the lenticular nucleus (LN) are characteristic transcranial sonography (TCS) echo features in patients with primary dystonia. This study aimed to explore alterations in the basal ganglia in different forms of primary focal dystonia.</p><p><b>METHODS</b>cross-sectional observational study was performed between December 2013 and December 2014 in 80 patients with different forms of primary focal dystonia and 55 neurologically normal control subjects. TCS was performed in patients and control subjects. Multiple comparisons of multiple rates were used to compare LN hyperechogenicity ratios between control and patient groups.</p><p><b>RESULTS</b>Thirteen individuals were excluded due to poor temporal bone windows, and two subjects were excluded due to disagreement in evaluation by sonologists. Totally, 70 patients (cervical dystonia, n = 30; blepharospasm, n = 30; oromandibular dystonia, n = 10) and 50 normal controls were included in the final analysis. LN hyperechogenicity was observed in 51% (36/70) of patients with primary focal dystonia, compared with 12% (6/50) of controls (P < 0.001). Substantia nigra hyperechogenicity did not differ between the two groups. LN hyperechogenicity was observed in 73% (22/30) of patients with cervical dystonia, a greater prevalence than in patients with blepharospasm (33%, 10/30, P = 0.002) and oromandibular dystonia (40%, 4/10, P = 0.126). LN hyperechogenicity was more frequently observed in patients with cervical dystonia compared with controls (73% vs. 12%, P < 0.001); however, no significant difference was detected in patients with blepharospasm (33% vs. 12%, P = 0.021) or oromandibular dystonia (40% vs. 12%, P = 0.088).</p><p><b>CONCLUSIONS</b>LN hyperechogenicity is more frequently observed in patients with primary focal dystonia than in controls. It does not appear to be a characteristic TCS echo feature in patients with blepharospasm or oromandibular dystonia.</p>

Basal Protein Expression Is Associated With Worse Outcome and Trastuzamab Resistance in HER2+ Invasive Breast Cancer.

BACKGROUND: We investigated the effect of basal protein expression on trastuzamab response in patients with HER2-positive (HER2(+)) breast cancer who received trastuzamab (T) and in HER2(+) breast cancer cell lines. PATIENTS AND METHODS: Expression of cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor (EGFR) was evaluated after immunohistochemical staining in paraffin-embedded tissue of 97 patients with stage I to III HER2(+) breast cancer treated with chemotherapy/T. Groups with and without basal protein expression were compared with respect to clinicopathologic parameters and survival. We treated 4 cell lines (2 basal-HER2 [HCC1569, HCC1954] and 2 nonbasal HER2 [BT474, SKBR3]) each with vehicle, T 20 µg/mL, paclitaxel 0.01 µM (P), and T with P (T + P). Cell viability was assessed and HER2 pathway suppression was compared between groups using immunoblot analysis. Mammosphere formation was used to assess breast cancer stem cell properties. RESULTS: EGFR expression was significantly associated with cancer-specific survival (CSS) (P = .05). CK5/6 expression strongly correlated with overall and disease-free survival, and CSS (P = .03, P = .04, and P = .03, respectively). Statistical significance was maintained for EGFR and CK5/6 after adjustment for covariates. CK14 was not associated with survival. All cell lines expressed similar levels of HER2. T and P alone inhibited proliferation of nonbasal cell lines; T + P had an additive cytotoxic effect. Basal cells were resistant to T, P inhibited proliferation, but T + P had no additive cytotoxic effect on cell growth in basal cells. Immunoblot analysis showed a significant decrease in phosphorylated Akt levels after treatment with T or T + P in nonbasal cells but not in basal cells. Akt blockade suppressed growth of basal and nonbasal HER2(+) cells. Furthermore, basal HER2 cell lines had increased mammosphere formation, which suggests increased stem cell properties compared with nonbasal HER2 cell lines. CONCLUSION: CK5/6 and EGFR expression are predictive of worse prognosis in HER2(+) breast cancer patients treated with T. Basal HER2 breast cancer cell lines are resistant to trastuzamab, which is mediated through the Akt pathway; AKT inhibition abrogates this resistance. Basal HER2 cell lines also have increased stem cell properties, which might play a role in the resistance pathway.

Progesterone and heparin-binding epidermal growth factor-like growth factor regulate the expression of tight junction protein Claudin-3 during early pregnancy.

OBJECTIVE: To determine Claudin-3 expression and its regulatory factors during embryo implantation. DESIGN: Experimental mouse models and cell culture. SETTING: University research laboratory. ANIMAL(S): Sexually mature female CD-1 strain mice. INTERVENTION(S): Ovariectomy and treatments. MAIN OUTCOME MEASURE(S): In situ hybridization and immunohistochemistry for detecting Claudin-3 messenger RNA and protein expression in mouse uterus, respectively; Western blot for detecting protein levels; immunofluorescence for detecting Claudin-3 protein in cultured cells. RESULT(S): Claudin-3 is strongly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy, and diminished at day 5 implantation sites. Then it is expressed at secondary decidual zone on day 8. Pseudopregnant uteri have a similar expression pattern as pregnant uteri from days 1-5. Claudin-3 expression is down-regulated after delayed implantation is activated by estrogen (E) treatment. Meanwhile Claudin-3 expression is stimulated by artificial decidualization. In ovariectomized mice, P induces Claudin-3 expression in the luminal epithelium, which is abrogated by P receptor antagonist RU486. Heparin-binding-epidermal growth factor (HB-EGF) down-regulates Claudin-3 expression, but enhances transcription factor Snail expression. In human endometrial epithelial ECC-1 cells, both E and P could stimulate Claudin-3 expression, whereas HB-EGF decreases Claudin-3 and increases Snail expression. CONCLUSION(S): Claudin-3 expression in uterine luminal epithelium is stimulated by P and suppressed by HB-EGF in mice and humans.

A novel role of IGFBP7 in mouse uterus: regulating uterine receptivity through Th1/Th2 lymphocyte balance and decidualization.

Previously we have screened out Insulin-like Growth Factor Binding Protein 7 (IGFBP7) as a differentially expressed gene in post-implantation uterus versus pre-implantation uterus by suppressive subtractive hybridation. However its function in uterus was not clearly identified. In this research, the expression and function of IGFBP7 during post-implantation were studied. We found that IGFBP7 was mainly located in the glandular epithelium and the stroma, and was upregulated after embryo implantation. The vector pCR3.1-IGFBP7-t expressing partial IGFBP7 was constructed. Inhibition of IGFBP7 by specific DNA immunization induced significant reduction of implanted embryos and pregnancy rate. The number of implanted embryos (5.68 ± 0.46) was significantly reduced after immunization with pCR3.1-IGFBP7-t, as compared with that of the mice immunized with the control vector (12.29 ± 0.36) or saline (14.58 ± 0.40) (p<0.01). After specific inhibition of IGFBP7, the T helper type 1 (Th1) cytokine IFNγ, was significantly elevated (p<0.05) and the Th2 cytokines IL-4 and IL-10, were reduced in uteri (p<0.05). The increase of Tbet and the decrease of Gata3 were found in mice peripheral lymphocytes by flow cytometry. The expression of decidualization marker IGFBP1 and angiogenesis regulator VEGF were declined in uteri (p<0.05). The expression of apoptosis-associated proteins, caspase3 and Bcl-2, were also declined (p<0.05). These results showed that inhibition of IGFBP7 induced pregnancy failure by shifting uterine cytokines to Th1 type dominance and repressing uterine decidualization.

Retinoic acid synthesis and metabolism are concurrent in the mouse uterus during peri-implantation.

Vitamin A (retinol) and its active metabolite, retinoic acid (RA), serve dual roles in the female reproductive tract. Cytochrome P450 26A1 (Cyp26a1), an RA-metabolizing enzyme, is involved in mammalian early pregnancy. In order to investigate the role of RA synthesis and metabolism during embryo implantation, we first investigated the spatiotemporal expression of RA-signal in the mouse uterus during the peri-implantation period. RA-signal-related molecules, including binding proteins, synthesizing enzymes, catabolizing enzymes and receptors, were all expressed in the mouse uterus during embryo implantation. The locations of the RA synthetic system (Aldh1a1, Aldh1a2, CRBP1) and catabolizing enzyme (Cyp26a1) were distinctive in the mouse uterus during the peri-implantation period. Aldh1a1 was located in the gland epithelium, whereas Aldh1a2 and CRBP1 were located in the stroma and Cyp26a1 was expressed in the luminal and glandular epithelium. These results demonstrate that RA synthesis occurs in the stroma, whereas RA metabolism takes place in the endometrial epithelium. When endometrial epithelial cells were isolated on day 4.5 of pregnancy and treated with E(2) (17beta-estradiol) or a combination of E(2) and progesterone, all-trans-RA (10 µM) significantly down-regulated the expression of LIF, HB-EF and CSF-1 in these cells in vitro. Taken together, these results suggest that the accumulation of RA in the stroma during mouse embryo implantation has an inhibitory effect on the expression of the three implantation-essential genes, LIF, HB-EGF and CSF-1. Therefore, the expression of Cyp26a1 in luminal and glandular epithelium might block the adverse effect of RA in order to promote successful embryo implantation.

Estradiol-regulated proline-rich acid protein 1 is repressed by class I histone deacetylase and functions in peri-implantation mouse uterus.

Secretory protein proline-rich acid protein 1 (PRAP1) is abundantly expressed in late pregnant uterus. However, regulation and function of PRAP1 in pregnant uterus is still elusive. We firstly reported differential expression of PRAP1 in peri-implantation uteri and its localization in endometrial epithelia. Expression of PRAP1 in uterus was induced by 17ß-estradiol and its expression showed a negative correlation with that of class Ihistone deacetylases (HDACs) in isolated endometrial epithelia. PRAP1 was increased by HDACs inhibitor sodium butyrate treatment, while decreased significantly by estrogen receptor inhibitor ICI182,780 via up-regulating class IHDACs. Number of implanted embryos was decreased in mice immunized with pCR3.1-PRAP1 or injected with rabbit anti-PRAP1 antibody. DNA immunization or antibody injection could affect apoptosis and expression of cytokines (IL-4, IFN-γ). In conclusion, both 17ß-estradiol and class IHDACs are involved in modulating PRAP1 expression in peri-implantation uteri. Preliminary functional research indicates that neutralizing PRAP1 protein causes reduction of implanted embryos.

Role of secretory protease inhibitor SPINK3 in mouse uterus during early pregnancy.

Successful embryo implantation depends on intricate epithelial-stromal cross-talk. However, molecular modulators involved in this cellular communication remain poorly elucidated. Using multiple approaches, we have investigated the spatiotemporal expression and regulation of serine protease inhibitor Kazal type 3 (SPINK3) in mouse uterus during the estrous cycle and early pregnancy. In cycling mice, both SPINK3 mRNA and protein are only expressed during proestrus. In the pregnant mouse, the expression levels of both SPINK3 mRNA and protein increase on days 5-8 and then decline. Spink3 mRNA is expressed exclusively in the uterine glandular epithelium, whereas SPINK3 protein is localized on the surface of both luminal and glandular epithelium and in the decidua. Moreover, SPINK3 in the decidua has been observed in the primary decidual zone on day 6 and the secondary decidual zone on days 7-8; this is tightly associated with the progression of decidualization. SPINK3 has also been found in decidual cells of the artificially decidualized uterine horn but not control horn, whereas Spink3 mRNA localizes in the glands of both horns. The expression of endometrial Spink3 is not regulated by the blastocyst according to its expression pattern during pseudopregnancy and delayed implantation but is induced by progesterone and further augmented by a combination of progesterone and estrogen in ovariectomized mice. Thus, uterine-gland-derived SPINK3, as a new paracrine modulator, might play an important role in embryo implantation through its influence on stromal decidualization in mice.

Retinoic acid-metabolizing enzyme cytochrome P450 26a1 (cyp26a1) is essential for implantation: functional study of its role in early pregnancy.

Vitamin A (VA) is required for normal fetal development and successful pregnancy. Excessive VA intake during pregnancy may lead to adverse maternal and fetal effects. Cytochrome P450 26A1 (cyp26a1), a retinoic acid (RA)-metabolizing enzyme, is involved in VA metabolism. It has been shown that cyp26a1 is expressed in female reproductive tract, especially in uterus. In order to investigate the role of cyp26a1 during pregnancy, we constructed a recombinant plasmid DNA vaccine encoding cyp26a1 protein and immunized mice with the plasmid. Compared to control groups, the pregnancy rate of the cyp26a1 plasmid-immunized mice were significantly decreased (P < 0.01). Further results showed that both cyp26a1 mRNA and protein were specifically induced in the uterus during implantation period and localized in the uterine luminal epithelium. Importantly, the number of implantation sites was also significantly reduced (P < 0.05) after the uterine injection of cyp26a1-specific antisense oligos or anti-cyp26a1 antibody on day 3 of pregnancy. Accordingly, the expression of RA-related cellular retinoic acid binding protein 1 and tissue transglutaminase was markedly increased (P < 0.05) in the uterine luminal epithelium after intrauterine injection treatments. These data demonstrate that uterine cyp26a1 activity is important for the maintenance of pregnancy, especially during the process of blastocyst implantation.