Massively parallel assessment of human variants with base editor screens.

Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.

Automation of absolute protein-ligand binding free energy calculations for docking refinement and compound evaluation.

Absolute binding free energy calculations with explicit solvent molecular simulations can provide estimates of protein-ligand affinities, and thus reduce the time and costs needed to find new drug candidates. However, these calculations can be complex to implement and perform. Here, we introduce the software BAT.py, a Python tool that invokes the AMBER simulation package to automate the calculation of binding free energies for a protein with a series of ligands. The software supports the attach-pull-release (APR) and double decoupling (DD) binding free energy methods, as well as the simultaneous decoupling-recoupling (SDR) method, a variant of double decoupling that avoids numerical artifacts associated with charged ligands. We report encouraging initial test applications of this software both to re-rank docked poses and to estimate overall binding free energies. We also show that it is practical to carry out these calculations cheaply by using graphical processing units in common machines that can be built for this purpose. The combination of automation and low cost positions this procedure to be applied in a relatively high-throughput mode and thus stands to enable new applications in early-stage drug discovery.

Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer's disease mouse model.

There is increasing evidence that inducing neuronal mitophagy can be used as a therapeutic intervention for Alzheimer's disease. Here, we screen a library of 2024 FDA-approved drugs or drug candidates, revealing UMI-77 as an unexpected mitophagy activator. UMI-77 is an established BH3-mimetic for MCL-1 and was developed to induce apoptosis in cancer cells. We found that at sub-lethal doses, UMI-77 potently induces mitophagy, independent of apoptosis. Our mechanistic studies discovered that MCL-1 is a mitophagy receptor and directly binds to LC3A. Finally, we found that UMI-77 can induce mitophagy in vivo and that it effectively reverses molecular and behavioral phenotypes in the APP/PS1 mouse model of Alzheimer's disease. Our findings shed light on the mechanisms of mitophagy, reveal that MCL-1 is a mitophagy receptor that can be targeted to induce mitophagy, and identify MCL-1 as a drug target for therapeutic intervention in Alzheimer's disease.

Assessment of the Diagnostic Potential of miR-29a-3p and miR-92a-3p as Circulatory Biomarkers in Acute Myeloid Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a set of Myeloproliferative neoplasms that are identified by excessive growth of myeloid blasts and production of abnormal blood cells. AML is the most common type of acute leukemia that occurs in adults. In addition, AML progresses rapidly and is considered a fatal disease. Thus, there is an urgent need to find new targets for molecularly designed therapies. In This study, we evaluated the circulatory levels of microRNA-29a-3p (miR-29a-3p) and miR-92a-3p beside exploring the expression pattern of their target gene myeloid cell leukemia sequence1 (MCL1) to investigate the role of these molecules in AML pathophysiology and to assess their ability to diagnose AML patients. METHODS: 40 adult AML patients along with 20 healthy subjects were enrolled in this study. Plasma were separated from venous blood samples, collected on EDTA, of all individuals were used to assess circulating miRNAs' levels. In the meantime, total RNA was extracted from isolated leukocytes and was used to quantify target mRNA transcript levels. RESULTS: Our data revealed that the circulating levels of miR-29a-3p and miR-92a-3p exhibited significant reduction in 90% and 100% of AML patients, respectively, when compared to the control group (p<0.001). On the other hand, the transcript level of the target gene of these miRNAs, MCL1, showed a sharp increase in 77.5% (p<0.001) of AML patients, along with a negative correlation with its regulatory miRNAs, miR-29a-3p and miR-92a-3p. CONCLUSION: Our data validates the negative regulatory role of miR-29a-3p and miR-92a-3p to the expression levels of MCL1 in peripheral blood and indicates that these miRNAs can be used as non-invasive diagnostic markers. Furthermore, our study highlights the therapeutic potential of miR-29a-3p and miR-92a-3p to target and downregulate a very important gene (MCL1), which is highly implicated in the pathogenesis of AML.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

In Vitro Assessment of the Anticancer Potential of Evodiamine in Human Oral Cancer Cell Lines.

Evodiamine, a bioactive alkaloid, has been regarded as having antioxidant, antiinflammatory, and anticancer properties. In the present study, we explored the effects of evodiamine on cell growth and apoptosis in human oral cancer cell lines. Our data revealed that evodiamine significantly inhibited the proliferation of human oral cancer cells and resulted in the cleavages of PARP (poly (ADP-ribose) polymerase) and caspase-3, in addition to causing the typical characteristics of apoptosis. Evodiamine also increased Bax protein levels and caused translocation of Bax into mitochondria and Bax oligomerization. In addition, evodiamine decreased expression of myeloid cell leukemia (Mcl-1) at the transcriptional modification, and knockdown of Mcl-1 clearly resulted in an increase in expression of Bax and active Bax, resulting in induction of apoptosis. Evodiamine reduced expression of phosphorylated AKT, and LY294002 potentiated evodiamine-induced apoptosis by regulating Mcl-1 protein. Our results suggest that evodiamine induces apoptosis in human oral cancer cells through the AKT pathway. These findings provide a rationale for its clinical application in the treatment of oral cancer.

Assessment of ABT-263 activity across a cancer cell line collection leads to a potent combination therapy for small-cell lung cancer.

BH3 mimetics such as ABT-263 induce apoptosis in a subset of cancer models. However, these drugs have shown limited clinical efficacy as single agents in small-cell lung cancer (SCLC) and other solid tumor malignancies, and rational combination strategies remain underexplored. To develop a novel therapeutic approach, we examined the efficacy of ABT-263 across >500 cancer cell lines, including 311 for which we had matched expression data for select genes. We found that high expression of the proapoptotic gene Bcl2-interacting mediator of cell death (BIM) predicts sensitivity to ABT-263. In particular, SCLC cell lines possessed greater BIM transcript levels than most other solid tumors and are among the most sensitive to ABT-263. However, a subset of relatively resistant SCLC cell lines has concomitant high expression of the antiapoptotic myeloid cell leukemia 1 (MCL-1). Whereas ABT-263 released BIM from complexes with BCL-2 and BCL-XL, high expression of MCL-1 sequestered BIM released from BCL-2 and BCL-XL, thereby abrogating apoptosis. We found that SCLCs were sensitized to ABT-263 via TORC1/2 inhibition, which led to reduced MCL-1 protein levels, thereby facilitating BIM-mediated apoptosis. AZD8055 and ABT-263 together induced marked apoptosis in vitro, as well as tumor regressions in multiple SCLC xenograft models. In a Tp53; Rb1 deletion genetically engineered mouse model of SCLC, the combination of ABT-263 and AZD8055 significantly repressed tumor growth and induced tumor regressions compared with either drug alone. Furthermore, in a SCLC patient-derived xenograft model that was resistant to ABT-263 alone, the addition of AZD8055 induced potent tumor regression. Therefore, addition of a TORC1/2 inhibitor offers a therapeutic strategy to markedly improve ABT-263 activity in SCLC.

Evaluation and critical assessment of putative MCL-1 inhibitors.

High levels of BCL-2 family proteins are implicated in a failed/ineffective apoptotic programme, often resulting in diseases, including cancer. Owing to their potential as drug targets in cancer therapy, several inhibitors of BCL-2 family proteins have been developed. These primarily target specific members of the BCL-2 family, particularly BCL-2 and BCL-XL but are ineffective against MCL-1. Major efforts have been invested in developing inhibitors of MCL-1, which is commonly amplified in human tumours and associated with tumour relapse and chemoresistance. In this report, the specificity of several BCL-2 family inhibitors (ABT-263, UCB-1350883, apogossypol and BH3I-1) was investigated and compared with putative MCL-1 inhibitors designed to exhibit improved or selective binding affinities for MCL-1 (TW-37, BI97C1, BI97C10, BI112D1, compounds 6 and 7, and MCL-1 inhibitor molecule (MIM-1)). ABT-263, BI97C1, BI112D1, MIM-1 and TW-37 exhibited specificity in inducing apoptosis in a Bax/Bak- and caspase-9-dependent manner, whereas the other agents showed no killing activity, or little or no specificity. Of these inhibitors, only ABT-263 and UCB-1350883 induced apoptosis in a BCL-2- or BCL-XL-dependent system. In cells that depend on MCL-1 for survival, ABT-263 and TW-37 induced extensive apoptosis, suggesting that at high concentrations these inhibitors have the propensity to inhibit MCL-1 in a cellular context. TW-37 induced apoptosis, assessed by chromatin condensation, caspase processing and phosphatidylserine externalisation, in a BAK-dependent manner and in cells that require MCL-1 for survival. TW-37-mediated apoptosis was also partly dependent on NOXA, suggesting that derivatives of TW-37, if engineered to exhibit better selectivity and efficacy at low nanomolar concentrations, may provide useful lead compounds for further synthetic programmes. Expanded medicinal chemistry iteration, as performed for the ABT series, may likewise improve the potency and specificity of the evaluated MCL-1 inhibitors.

Prognostic assessment of three single-nucleotide polymorphisms (GNB3 825C>T, BCL2-938C>A, MCL1-386C>G) in extrahepatic cholangiocarcinoma.

BACKGROUND/AIMS: Cholangiocellular carcinoma (CCA) has a devastating prognosis and markers enabling a precise prediction of the clinical outcome have long remained scarce. Recently, it has been demonstrated that genotype distribution of several single-nucleotide polymorphisms (SNPs) in genes that modulate G protein-signal transduction and apoptosis can serve as helpful predictive parameters in various carcinomas. We here aimed at extending the panel of SNPs suitable for predicting the outcome of CCA. METHODOLOGY: Forty Caucasian patients with extrahepatic CCA and 40 age- and sex-matched healthy white Caucasians were genotyped to elucidate putative associations between clinical outcome and genotypes of the three following SNPs: G protein beta 3 (GNB3) 825C>T, B-cell-lymphoma-2 (Bcl-2) -938C>A, and myeloid cell leukemia-1 (Mcl-1) -386C>G. RESULTS: Patients homozygous for the C allele of the GNB3 825C>T polymorphism exhibited a significant prolonged overall survival compared with patients displaying the CT or TT genotype (median survival [months]: 31 vs. 13 vs. 7; p < .05) and also showed lower bilirubin serum levels. Additionally, the CC genotype of the BCL2-938C>A polymorphism was associated with higher GLDH serum activities (U/l; 29.8 +/- 7.1 vs. 11.4 +/- 4.3 vs. 5.6 +/- 1.7 comparing CC vs. CA vs. AA; p < .05). Genotype distributions for all SNPs were not significantly different in patients vs. controls. CONCLUSIONS: GNB3 825C>T SNP may be a novel independent prognostic marker for patients suffering from extrahepatic CCA with the CC genotype to be associated with a favorable clinical outcome. Further prospective studies are needed to confirm these results and reveal additional functional SNP effects.