Primary acute lymphoblastic leukemia cells are susceptible to microtubule depolymerization in G1 and M phases through distinct cell death pathways.

Microtubule targeting agents (MTAs) are widely used cancer chemotherapeutics which conventionally exert their effects during mitosis, leading to mitotic or postmitotic death. However, accumulating evidence suggests that MTAs can also generate death signals during interphase, which may represent a key mechanism in the clinical setting. We reported previously that vincristine and other microtubule destabilizers induce death not only in M phase but also in G1 phase in primary acute lymphoblastic leukemia cells. Here, we sought to investigate and compare the pathways responsible for phase-specific cell death. Primary acute lymphoblastic leukemia cells were subjected to centrifugal elutriation, and cell populations enriched in G1 phase (97%) or G2/M phases (80%) were obtained and treated with vincristine. We found death of M phase cells was associated with established features of mitochondrial-mediated apoptosis, including Bax activation, loss of mitochondrial transmembrane potential, caspase-3 activation, and nucleosomal DNA fragmentation. In contrast, death of G1 phase cells was not associated with pronounced Bax or caspase-3 activation but was associated with loss of mitochondrial transmembrane potential, parylation, nuclear translocation of apoptosis-inducing factor and endonuclease G, and supra-nucleosomal DNA fragmentation, which was enhanced by inhibition of autophagy. The results indicate that microtubule depolymerization induces distinct cell death pathways depending on during which phase of the cell cycle microtubule perturbation occurs. The observation that a specific type of drug can enter a single cell type and induce two different modes of death is novel and intriguing. These findings provide a basis for advancing knowledge of clinical mechanisms of MTAs.

Synthesis and Anti-Melanoma Activity of Acryloyl Pyridinone Analogues.

A major challenge for clinical management of melanoma is the prevention and treatment of metastatic disease. Drug discovery efforts over the last 10 years have resulted in several drugs that improve the prognosis of metastatic melanoma; however, most patients develop early resistance to these treatments. We designed and synthesized, through a concise synthetic strategy, a series of hybrid olefin-pyridinone compounds that consist of structural motifs from tamoxifen and ilicicolin H. These compounds were tested against a human melanoma cell line and patient-derived melanoma cells that had metastasized to the brain. Three compounds 7 b, 7 c, and 7 g demonstrated promising activity (IC50=0.4-4.3 µM). Cell cycle analysis demonstrated that 7 b and 7 c induce cell cycle arrest predominantly in the G1 phase. Both 7 b and 7c significantly inhibited migration of A375 melanoma cells; greater effects were demonstrated by 7 b. Molecular modelling analysis provides insight into a plausible mechanism of action.

CRISPR/dCas9-KRAB-Mediated Suppression of S100b Restores p53-Mediated Apoptosis in Melanoma Cells.

Overexpression of S100B is routinely used for disease-staging and for determining prognostic outcomes in patients with malignant melanoma. Intracellular interactions between S100B and wild-type (WT)-p53 have been demonstrated to limit the availability of free WT-p53 in tumor cells, inhibiting the apoptotic signaling cascade. Herein, we demonstrate that, while oncogenic overexpression of S100B is poorly correlated (R < 0.3; p > 0.05) to alterations in S100B copy number or DNA methylation in primary patient samples, the transcriptional start site and upstream promoter of the gene are epigenetically primed in melanoma cells with predicted enrichment of activating transcription factors. Considering the regulatory role of activating transcription factors in S100B upregulation in melanoma, we stably suppressed S100b (murine ortholog) by using a catalytically inactive Cas9 (dCas9) fused to a transcriptional repressor, Krüppel-associated box (KRAB). Selective combination of S100b-specific single-guide RNAs and the dCas9-KRAB fusion significantly suppressed expression of S100b in murine B16 melanoma cells without noticeable off-target effects. S100b suppression resulted in recovery of intracellular WT-p53 and p21 levels and concomitant induction of apoptotic signaling. Expression levels of apoptogenic factors (i.e., apoptosis-inducing factor, caspase-3, and poly-ADP ribose polymerase) were altered in response to S100b suppression. S100b-suppressed cells also showed reduced cell viability and increased susceptibility to the chemotherapeutic agents, cisplatin and tunicamycin. Targeted suppression of S100b therefore offers a therapeutic vulnerability to overcome drug resistance in melanoma.

The mycelium of the Trametes versicolor synn. Coriolus versicolor (Turkey tail mushroom) exhibit anti-melanoma activity in vitro.

Melanoma is one of the most aggressive forms of skin cancer and is characterized by high metastatic potential. Despite improvements in early diagnosis and treatment, the mortality rate among metastatic melanoma patients continues to represent a significant clinical challenge. Therefore, it is imperative that we search for new forms of treatment. Trametes versicolor is a mushroom commonly used in Chinese traditional medicine due to its numerous beneficial properties. In the present work, we demonstrate T. versicolor fruiting body and mycelium ethanol extracts exhibit potent cytotoxic activity towards A375 (IC50 = 663.3 and 114.5 µg/mL respectively) and SK-MEL-5 (IC50 = 358.4 and 88.6 µg/mL respectively) human melanoma cell lines. Further studies revealed that T. versicolor mycelium extract induced apoptotic cell death and poly (ADP-ribose) polymerase cleavage, upregulated the expression of autophagy-associated marker LC3-II, increased the presentation of major histocompatibility complex II and expression of programmed death-ligand receptor, and inhibited cell migration in SK-MEL-5 cells. Therefore, our present findings highlight the therapeutic potential of T. versicolor mycelium extract for the treatment of melanoma and merit further study.

Resveratrol induces major histocompatibility complex class I antigen presentation in a STING-dependent and independent manner in melanoma.

Immune checkpoint inhibitor therapy has drastically improved outcomes in treating cancer, particularly in melanoma. However, half of melanoma patients are resistant to treatment. One mechanism used by tumor cells to evade immune attack is to down-regulate major histocompatibility complex (MHC) class I molecules, which are required for cytotoxic CD8 T-cells to eliminate cancer cells. To increase immunotherapeutic efficacy, it is critical to identify how to restore MHC-I expression on cancer cells so that tumor antigens are presented. We found that resveratrol elevated MHC-I expression, so that tumor antigens are presented to cytotoxic CD8 T-cell killing. Through proteomic interrogation, we identified the STING pathway as a potential mechanism of action. Further studies indicated that resveratrol-mediated regulation of STING induced MHC-I expression potentially through both interferon-independent and dependent pathways. Our results have indicated the potential of STING to induce MHC-I expression independent of interferon signaling, broadening the potential of STING modulation as a tool to improve immune checkpoint blockade.

Anti-glioblastoma activity of monensin and its analogs in an organoid model of cancer.

Glioblastoma (GBM) remains the most frequently diagnosed primary malignant brain cancer in adults. Despite recent progress in understanding the biology of GBM, the clinical outcome for patients remains poor, with a median survival of approximately one year after diagnosis. One factor contributing to failure in clinical trials is the fact that traditional models used in GBM drug discovery poorly recapitulate patient tumors. Previous studies have shown that monensin (MON) analogs, namely esters and amides on C-26 were potent towards various types of cancer cell lines. In the present study we have investigated the activity of these molecules in GBM organoids, as well as in a host:tumor organoid model. Using a mini-ring cell viability assay we have identified seven analogs (IC50 = 91.5 ± 54.4-291.7 ± 68.8 nM) more potent than parent MON (IC50 = 612.6 ± 184.4 nM). Five of these compounds induced substantial DNA fragmentation in GBM organoids, suggestive of apoptotic cell death. The most active analog, compound 1, significantly reduced GBM cell migration, induced PARP degradation, diminished phosphorylation of STAT3, Akt and GSK3ß, increased É£H2AX signaling and upregulated expression of the autophagy associated marker LC3-II. To investigate the activity of MON and compound 1 in a tumor microenvironment, we developed human cerebral organoids (COs) from human induced pluripotent stem cells (iPSCs). The COs showed features of early developing brain such as multiple neural rosettes with a proliferative zone of neural stem cells (Nestin+), neurons (TUJ1 +), primitive ventricular system (SOX2 +/Ki67 +), intermediate zone (TBR2 +) and cortical plate (MAP2 +). In order to generate host:tumor organoids, we co-cultured RFP-labeled U87MG cells with fully formed COs. Compound 1 and MON reduced U87MG tumor size in the COs after four days of treatment and induced a significant reduction of PARP expression. These findings highlight the therapeutic potential of MON analogs towards GBM and support the application of organoid models in anti-cancer drug discovery.

Phosphoproteomics Provides Novel Insights into the Response of Primary Acute Lymphoblastic Leukemia Cells to Microtubule Depolymerization in G1 Phase of the Cell Cycle.

Microtubule targeting agents (MTAs) have been used for the treatment of cancer for many decades and are among the most successful chemotherapeutic agents. However, their application and effectiveness are limited because of toxicity and resistance as well as a lack of knowledge of molecular mechanisms downstream of microtubule inhibition. Insights into key pathways that link microtubule disruption to cell death is critical for optimal use of these drugs, for defining biomarkers useful in patient stratification, and for informed design of drug combinations. Although MTAs characteristically induce death in mitosis, microtubule destabilizing agents such as vincristine also induce death directly in G1 phase in primary acute lymphoblastic leukemia (ALL) cells. Because many signaling pathways regulating cell survival and death involve changes in protein expression and phosphorylation, we undertook a comprehensive quantitative proteomic study of G1 phase ALL cells treated with vincristine. The results revealed distinct alterations associated with c-Jun N-terminal kinase signaling, anti-proliferative signaling, the DNA damage response, and cytoskeletal remodeling. Signals specifically associated with cell death were identified by pre-treatment with the CDK4/6 inhibitor palbociclib, which caused G1 arrest and precluded death induction. These results provide insights into signaling mechanisms regulating cellular responses to microtubule inhibition and provide a foundation for a better understanding of the clinical mechanisms of MTAs and for the design of novel drug combinations. The mass spectrometry proteomics data have been deposited to the PRIDE Archive (http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD027190 and 10.6019/PXD027190.