Genetic ablation of Immt induces a lethal disruption of the MICOS complex.

The mitochondrial contact site and cristae organizing system (MICOS) is important for crista junction formation and for maintaining inner mitochondrial membrane architecture. A key component of the MICOS complex is MIC60, which has been well studied in yeast and cell culture models. However, only one recent study has demonstrated the embryonic lethality of losing Immt (the gene encoding MIC60) expression. Tamoxifen-inducible ROSA-CreERT2-mediated deletion of Immt in adult mice disrupted the MICOS complex, increased mitochondria size, altered cristae morphology, and was lethal within 12 d. Pathologically, these mice displayed defective intestinal muscle function (paralytic ileus) culminating in dehydration. We also identified bone marrow (BM) hypocellularity in Immt-deleted mice, although BM transplants from wild-type mice did not improve survival. Altogether, this inducible mouse model demonstrates the importance of MIC60 in vivo, in both hematopoietic and non-hematopoietic tissues, and provides a valuable resource for future mechanistic investigations into the MICOS complex.

A 3D Perfusable Platform for In Vitro Culture of Patient Derived Xenografts.

Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.

Genetic loci associated with skin pigmentation in African Americans and their effects on vitamin D deficiency.

A recent genome-wide association study (GWAS) in African descent populations identified novel loci associated with skin pigmentation. However, how genomic variations affect skin pigmentation and how these skin pigmentation gene variants affect serum 25(OH) vitamin D variation has not been explored in African Americans (AAs). In order to further understand genetic factors that affect human skin pigmentation and serum 25(OH)D variation, we performed a GWAS for skin pigmentation with 395 AAs and a replication study with 681 AAs. Then, we tested if the identified variants are associated with serum 25(OH) D concentrations in a subset of AAs (n = 591). Skin pigmentation, Melanin Index (M-Index), was measured using a narrow-band reflectometer. Multiple regression analysis was performed to identify variants associated with M-Index and to assess their role in serum 25(OH)D variation adjusting for population stratification and relevant confounding variables. A variant near the SLC24A5 gene (rs2675345) showed the strongest signal of association with M-Index (P = 4.0 x 10-30 in the pooled dataset). Variants in SLC24A5, SLC45A2 and OCA2 together account for a large proportion of skin pigmentation variance (11%). The effects of these variants on M-Index was modified by sex (P for interaction = 0.009). However, West African Ancestry (WAA) also accounts for a large proportion of M-Index variance (23%). M-Index also varies among AAs with high WAA and high Genetic Score calculated from top variants associated with M-Index, suggesting that other unknown genomic factors related to WAA are likely contributing to skin pigmentation variation. M-Index was not associated with serum 25(OH)D concentrations, but the Genetic Score was significantly associated with vitamin D deficiency (serum 25(OH)D levels less than 12 ng/mL) (OR, 1.30; 95% CI, 1.04-1.64). The findings support the hypothesis suggesting that skin pigmentation evolved responding to increased demand for subcutaneous vitamin D synthesis in high latitude environments.

Genetic Ancestry Analysis Reveals Misclassification of Commonly Used Cancer Cell Lines.

BACKGROUND: Given the scarcity of cell lines from underrepresented populations, it is imperative that genetic ancestry for these cell lines is characterized. Consequences of cell line mischaracterization include squandered resources and publication retractions. METHODS: We calculated genetic ancestry proportions for 15 cell lines to assess the accuracy of previous race/ethnicity classification and determine previously unknown estimates. DNA was extracted from cell lines and genotyped for ancestry informative markers representing West African (WA), Native American (NA), and European (EUR) ancestry. RESULTS: Of the cell lines tested, all previously classified as White/Caucasian were accurately described with mean EUR ancestry proportions of 97%. Cell lines previously classified as Black/African American were not always accurately described. For instance, the 22Rv1 prostate cancer cell line was recently found to carry mixed genetic ancestry using a much smaller panel of markers. However, our more comprehensive analysis determined the 22Rv1 cell line carries 99% EUR ancestry. Most notably, the E006AA-hT prostate cancer cell line, classified as African American, was found to carry 92% EUR ancestry. We also determined the MDA-MB-468 breast cancer cell line carries 23% NA ancestry, suggesting possible Afro-Hispanic/Latina ancestry. CONCLUSIONS: Our results suggest predominantly EUR ancestry for the White/Caucasian-designated cell lines, yet high variance in ancestry for the Black/African American-designated cell lines. In addition, we revealed an extreme misclassification of the E006AA-hT cell line. IMPACT: Genetic ancestry estimates offer more sophisticated characterization leading to better contextualization of findings. Ancestry estimates should be provided for all cell lines to avoid erroneous conclusions in disparities literature.

Defining specificity and on-target activity of BH3-mimetics using engineered B-ALL cell lines.

One of the hallmarks of cancer is a resistance to the induction of programmed cell death that is mediated by selection of cells with elevated expression of anti-apoptotic members of the BCL-2 family. To counter this resistance, new therapeutic agents known as BH3-mimetic small molecules are in development with the goal of antagonizing the function of anti-apoptotic molecules and promoting the induction of apoptosis. To facilitate the testing and modeling of BH3-mimetic agents, we have developed a powerful system for evaluation and screening of agents both in culture and in immune competent animal models by engineering mouse leukemic cells and re-programming them to be dependent on exogenously expressed human anti-apoptotic BCL-2 family members. Here we demonstrate that this panel of cell lines can determine the specificity of BH3-mimetics to individual anti-apoptotic BCL-2 family members (BCL-2, BCL-XL, BCL-W, BFL-1, and MCL-1), demonstrate whether cell death is due to the induction of apoptosis (BAX and BAK-dependent), and faithfully assess the efficacy of BH3-mimetic small molecules in pre-clinical mouse models. These cells represent a robust and valuable pre-clinical screening tool for validating the efficacy, selectivity, and on-target action of BH3-mimetic agents.

Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction.

MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity.

Cyclic GMP/protein kinase G type-Iα (PKG-Iα) signaling pathway promotes CREB phosphorylation and maintains higher c-IAP1, livin, survivin, and Mcl-1 expression and the inhibition of PKG-Iα kinase activity synergizes with cisplatin in non-small cell lung cancer cells.

Previously, our laboratory showed that nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G type-Iα (PKG-Iα) signaling pathway plays an important role in preventing spontaneous apoptosis and promoting cell proliferation in both normal cells (bone marrow stromal cells and vascular smooth muscle cells) and certain cancer cells (ovarian cancer cells). In the present study, we investigated the novel role of the cGMP/PKG-Iα pathway in preventing spontaneous apoptosis, promoting colony formation and regulating phosphorylation of cAMP response element binding (CREB) protein and protein expression of inhibitor of apoptosis proteins (IAPs) and anti-apoptotic Bcl-2-related proteins in NCI-H460 and A549 non-small cell lung cancer (NSCLC) cells. 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), which blocks endogenous NO-induced activation of cGMP/PKG-Iα, induced apoptosis and decreased colony formation. ODQ also decreased CREB ser133 phosphorylation and protein expression of c-IAP1, livin, and survivin. DT-2 (inhibitor of PKG-Iα kinase activity) increased apoptosis by twofold and decreased CREB ser133 phosphorylation and c-IAP1, livin, and survivin expression. Gene knockdown of PKG-Iα expression using small-interfering RNA increased apoptosis and decreased CREB ser133 phosphorylation, and c-IAP1, livin, survivin, and Mcl-1 expression. Inhibition of PKG-Iα kinase activity with DT-2 dramatically enhanced pro-apoptotic effects of the chemotherapeutic agent cisplatin. Combined treatment of DT-2 and cisplatin increased apoptosis compared with cisplatin or DT-2 alone, showing a synergistic effect. The data suggest that the PKG-Iα kinase activity is necessary for maintaining higher levels of CREB phosphorylation at ser133 and protein expression of c-IAP1, livin, survivin, and Mcl-1, preventing spontaneous apoptosis and promoting colony formation in NSCLC cells, which may limit the effectiveness of chemotherapeutic agents like cisplatin.

Anti-apoptotic MCL-1 localizes to the mitochondrial matrix and couples mitochondrial fusion to respiration.

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.

Ubiquitin-independent degradation of antiapoptotic MCL-1.

Antiapoptotic myeloid cell leukemia 1 (MCL-1) is an essential modulator of survival during the development and maintenance of a variety of cell lineages. Its turnover, believed to be mediated by the ubiquitin-proteasome system, facilitates apoptosis induction in response to cellular stress. To investigate the contribution of ubiquitinylation in regulating murine MCL-1 turnover, we generated an MCL-1 mutant lacking the lysine residues required for ubiquitinylation (MCL-1(KR)). Here, we demonstrate that despite failing to be ubiquitinylated, the MCL-1(KR) protein is eliminated at a rate similar to that of wild-type MCL-1 under basal and stressed conditions. Moreover, the degradation of wild-type MCL-1 is not affected when ubiquitin-activating enzyme E1 activity is blocked. Likewise, both wild-type and MCL-1(KR) proteins are similarly degraded when expressed in primary lymphocytes. Supporting these findings, unmodified, in vitro-translated MCL-1 can be degraded in a cell-free system by the 20S proteasome. Taken together, these data demonstrate that MCL-1 degradation can occur independently of ubiquitinylation.

Delayed cytotoxic effects of methyl jasmonate and cis-jasmone induced apoptosis in prostate cancer cells.

Advanced prostate cancer cells are typically hormone independent, resistant to apoptosis and do not respond to chemotherapeutic agents. The ability of methyl jasmonate (MJ) and cis-jasmone (CJ) to inhibit growth in hormone independent prostate cancer cell lines, PC-3 and DU-145, was evaluated. CJ and MJ inhibited cell growth, induced cell cycle arrest and apoptosis. Detailed studies with the PC-3 cell line revealed that 2 mM CJ or MJ treatment resulted in caspase 3 activation and Tumor Necrosis Factor Receptor 1 (TNFR1) activation, all hallmarks of apoptosis. These phytochemicals could be useful in the management of advanced prostate cancer.