Identification and Pharmacological Targeting of Treatment-Resistant, Stem-like Breast Cancer Cells for Combination Therapy.

Tumors frequently harbor isogenic yet epigenetically distinct subpopulations of multi-potent cells with high tumor-initiating potential-often called Cancer Stem-Like Cells (CSLCs). These can display preferential resistance to standard-of-care chemotherapy. Single-cell analyses can help elucidate Master Regulator (MR) proteins responsible for governing the transcriptional state of these cells, thus revealing complementary dependencies that may be leveraged via combination therapy. Interrogation of single-cell RNA sequencing profiles from seven metastatic breast cancer patients, using perturbational profiles of clinically relevant drugs, identified drugs predicted to invert the activity of MR proteins governing the transcriptional state of chemoresistant CSLCs, which were then validated by CROP-seq assays. The top drug, the anthelmintic albendazole, depleted this subpopulation in vivo without noticeable cytotoxicity. Moreover, sequential cycles of albendazole and paclitaxel-a commonly used chemotherapeutic -displayed significant synergy in a patient-derived xenograft (PDX) from a TNBC patient, suggesting that network-based approaches can help develop mechanism-based combinatorial therapies targeting complementary subpopulations.

Network-based elucidation of colon cancer drug resistance mechanisms by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. Leveraging progress in proteomic technologies and network-based methodologies, we introduce Virtual Enrichment-based Signaling Protein-activity Analysis (VESPA)-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and use it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogating tumor-specific enzyme/substrate interactions accurately infers kinase and phosphatase activity, based on their substrate phosphorylation state, effectively accounting for signal crosstalk and sparse phosphoproteome coverage. The analysis elucidates time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring, experimentally confirmed by CRISPR knock-out assays, suggesting broad applicability to cancer and other diseases.

Elucidating Compound Mechanism of Action and Polypharmacology with a Large-scale Perturbational Profile Compendium.

The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have implemented an efficient integrative experimental and computational framework leveraging the systematic generation and analysis of drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Protein activity-based analyses revealed highly reproducible, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide polypharmacology map, characterization of MoA-related drug clusters and off-target effects, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins. The proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, could help support more systematic and quantitative approaches to precision oncology.

Systematic elucidation and pharmacological targeting of tumor-infiltrating regulatory T cell master regulators.

Due to their immunosuppressive role, tumor-infiltrating regulatory T cells (TI-Tregs) represent attractive immuno-oncology targets. Analysis of TI vs. peripheral Tregs (P-Tregs) from 36 patients, across four malignancies, identified 17 candidate master regulators (MRs) as mechanistic determinants of TI-Treg transcriptional state. Pooled CRISPR-Cas9 screening in vivo, using a chimeric hematopoietic stem cell transplant model, confirmed the essentiality of eight MRs in TI-Treg recruitment and/or retention without affecting other T cell subtypes, and targeting one of the most significant MRs (Trps1) by CRISPR KO significantly reduced ectopic tumor growth. Analysis of drugs capable of inverting TI-Treg MR activity identified low-dose gemcitabine as the top prediction. Indeed, gemcitabine treatment inhibited tumor growth in immunocompetent but not immunocompromised allografts, increased anti-PD-1 efficacy, and depleted MR-expressing TI-Tregs in vivo. This study provides key insight into Treg signaling, specifically in the context of cancer, and a generalizable strategy to systematically elucidate and target MR proteins in immunosuppressive subpopulations.

Network-based elucidation of colon cancer drug resistance by phosphoproteomic time-series analysis.

Aberrant signaling pathway activity is a hallmark of tumorigenesis and progression, which has guided targeted inhibitor design for over 30 years. Yet, adaptive resistance mechanisms, induced by rapid, context-specific signaling network rewiring, continue to challenge therapeutic efficacy. By leveraging progress in proteomic technologies and network-based methodologies, over the past decade, we developed VESPA-an algorithm designed to elucidate mechanisms of cell response and adaptation to drug perturbations-and used it to analyze 7-point phosphoproteomic time series from colorectal cancer cells treated with clinically-relevant inhibitors and control media. Interrogation of tumor-specific enzyme/substrate interactions accurately inferred kinase and phosphatase activity, based on their inferred substrate phosphorylation state, effectively accounting for signal cross-talk and sparse phosphoproteome coverage. The analysis elucidated time-dependent signaling pathway response to each drug perturbation and, more importantly, cell adaptive response and rewiring that was experimentally confirmed by CRISPRko assays, suggesting broad applicability to cancer and other diseases.

Nuclear microRNA-466c regulates Vegfa expression in response to hypoxia.

MicroRNAs are well characterized in their role in silencing gene expression by targeting 3´-UTR of mRNAs in cytoplasm. However, recent studies have shown that miRNAs have a role in the regulation of genes in the nucleus, where they are abundantly located. We show here that in mouse endothelial cell line (C166), nuclear microRNA miR-466c participates in the regulation of vascular endothelial growth factor a (Vegfa) gene expression in hypoxia. Upregulation of Vegfa expression in response to hypoxia was significantly compromised after removal of miR-466c with CRISPR-Cas9 genomic deletion. We identified a promoter-associated long non-coding RNA on mouse Vegfa promoter and show that miR-466c directly binds to this transcript to modulate Vegfa expression. Collectively, these observations suggest that miR-466c regulates Vegfa gene transcription in the nucleus by targeting the promoter, and expands on our understanding of the role of miRNAs well beyond their canonical role.

Intracerebral overexpression of miR-669c is protective in mouse ischemic stroke model by targeting MyD88 and inducing alternative microglial/macrophage activation.

BACKGROUND: Ischemic stroke is a devastating disease without a cure. The available treatments for ischemic stroke, thrombolysis by tissue plasminogen activator, and thrombectomy are suitable only to a fraction of patients and thus novel therapeutic approaches are urgently needed. The neuroinflammatory responses elicited secondary to the ischemic attack further aggravate the stroke-induced neuronal damage. It has been demonstrated that these responses are regulated at the level of non-coding RNAs, especially miRNAs. METHODS: We utilized lentiviral vectors to overexpress miR-669c in BV2 microglial cells in order to modulate their polarization. To detect whether the modulation of microglial activation by miR-669c provides protection in a mouse model of transient focal ischemic stroke, miR-669c overexpression was driven by a lentiviral vector injected into the striatum prior to induction of ischemic stroke. RESULTS: Here, we demonstrate that miR-669c-3p, a member of chromosome 2 miRNA cluster (C2MC), is induced upon hypoxic and excitotoxic conditions in vitro and in two different in vivo models of stroke. Rather than directly regulating the neuronal survival in vitro, miR-669c is capable of attenuating the microglial proinflammatory activation in vitro and inducing the expression of microglial alternative activation markers arginase 1 (Arg1), chitinase-like 3 (Ym1), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Intracerebral overexpression of miR-669c significantly decreased the ischemia-induced cell death and ameliorated the stroke-induced neurological deficits both at 1 and 3 days post injury (dpi). Albeit miR-669c overexpression failed to alter the overall Iba1 protein immunoreactivity, it significantly elevated Arg1 levels in the ischemic brain and increased colocalization of Arg1 and Iba1. Moreover, miR-669c overexpression under cerebral ischemia influenced several morphological characteristics of Iba1 positive cells. We further demonstrate the myeloid differentiation primary response gene 88 (MyD88) transcript as a direct target for miR-669c-3p in vitro and show reduced levels of MyD88 in miR-669c overexpressing ischemic brains in vivo. CONCLUSIONS: Collectively, our data provide the evidence that miR-669c-3p is protective in a mouse model of ischemic stroke through enhancement of the alternative microglial/macrophage activation and inhibition of MyD88 signaling. Our results accentuate the importance of controlling miRNA-regulated responses for the therapeutic benefit in conditions of stroke and neuroinflammation.

Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery.

Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.

Contribution of allelic imbalance to colorectal cancer.

Point mutations in cancer have been extensively studied but chromosomal gains and losses have been more challenging to interpret due to their unspecific nature. Here we examine high-resolution allelic imbalance (AI) landscape in 1699 colorectal cancers, 256 of which have been whole-genome sequenced (WGSed). The imbalances pinpoint 38 genes as plausible AI targets based on previous knowledge. Unbiased CRISPR-Cas9 knockout and activation screens identified in total 79 genes within AI peaks regulating cell growth. Genetic and functional data implicate loss of TP53 as a sufficient driver of AI. The WGS highlights an influence of copy number aberrations on the rate of detected somatic point mutations. Importantly, the data reveal several associations between AI target genes, suggesting a role for a network of lineage-determining transcription factors in colorectal tumorigenesis. Overall, the results unravel the contribution of AI in colorectal cancer and provide a plausible explanation why so few genes are commonly affected by point mutations in cancers.

Comprehensive evaluation of coding region point mutations in microsatellite-unstable colorectal cancer.

Microsatellite instability (MSI) leads to accumulation of an excessive number of mutations in the genome, mostly small insertions and deletions. MSI colorectal cancers (CRCs), however, also contain more point mutations than microsatellite-stable (MSS) tumors, yet they have not been as comprehensively studied. To identify candidate driver genes affected by point mutations in MSI CRC, we ranked genes based on mutation significance while correcting for replication timing and gene expression utilizing an algorithm, MutSigCV Somatic point mutation data from the exome kit-targeted area from 24 exome-sequenced sporadic MSI CRCs and respective normals, and 12 whole-genome-sequenced sporadic MSI CRCs and respective normals were utilized. The top 73 genes were validated in 93 additional MSI CRCs. The MutSigCV ranking identified several well-established MSI CRC driver genes and provided additional evidence for previously proposed CRC candidate genes as well as shortlisted genes that have to our knowledge not been linked to CRC before. Two genes, SMARCB1 and STK38L, were also functionally scrutinized, providing evidence of a tumorigenic role, for SMARCB1 mutations in particular.

Doxycycline modulates VEGF-A expression: Failure of doxycycline-inducible lentivirus shRNA vector to knockdown VEGF-A expression in transgenic mice.

Vascular endothelial growth factor-A (VEGF-A) is the master regulator of angiogenesis, vascular permeability and growth. However, its role in mature blood vessels is still not well understood. To better understand the role of VEGF-A in the adult vasculature, we generated a VEGF-A knockdown mouse model carrying a doxycycline (dox)-regulatable short hairpin RNA (shRNA) transgene, which silences VEGF-A. The aim was to find the critical level of VEGF-A reduction for vascular well-being in vivo. In vitro, the dox-inducible lentiviral shRNA vector decreased VEGF-A expression efficiently and dose-dependently in mouse endothelial cells and cardiomyocytes. In the generated transgenic mice plasma VEGF-A levels decreased shortly after the dox treatment but returned back to normal after two weeks. VEGF-A expression decreased shortly after the dox treatment only in some tissues. Surprisingly, increasing the dox exposure time and dose led to elevated VEGF-A expression in some tissues of both wildtype and knockdown mice, suggesting that dox itself has an effect on VEGF-A expression. When the effect of dox on VEGF-A levels was further tested in naïve/non-transduced cells, the dox administration led to a decreased VEGF-A expression in endothelial cells but to an increased expression in cardiomyocytes. In conclusion, the VEGF-A knockdown was achieved in a dox-regulatable fashion with a VEGF-A shRNA vector in vitro, but not in the knockdown mouse model in vivo. Dox itself was found to regulate VEGF-A expression explaining the unexpected results in mice. The effect of dox on VEGF-A levels might at least partly explain its previously reported beneficial effects on myocardial and brain ischemia. Also, this effect on VEGF-A should be taken into account in all studies using dox-regulated vectors.

Myc requires RhoA/SRF to reprogram glutamine metabolism.

RhoA regulates actin cytoskeleton but recent evidence suggest a role for this conserved Rho GTPase also in other cellular processes, including transcriptional control of cell proliferation and survival. Interestingy, loss of RhoA is synthetic lethal with oncogenic Myc, a master transcription factor that turns on anabolic metabolism to promote cell growth in many cancers. We show evidence indicating that the synthetic lethal interaction between RhoA loss and Myc arises from deficiency in glutamine utilization, resulting from impaired co-regulation of glutaminase expression and anaplerosis by Myc and RhoA - serum response factor (SRF) pathway. The results suggest metabolic coordination between Myc and RhoA/SRF in sustaining cancer cell viability and indicate RhoA/SRF as a potential vulnerability in cancer cells for therapeutic targeting.

Enhancing Angiogenesis in Mice by VEGF-Targeting Small Activating RNAs.

The prevalence of cardiovascular diseases is steadily increasing, and it is the leading cause of death worldwide. Therefore, new treatments, such as gene therapy are needed. During the last decade, the role of small noncoding RNAs (ncRNAs) in the regulation of gene expression at the transcriptional level has been shown. Promoter-targeted small RNAs recruit histone-modifying enzymes and can either repress or induce target gene expression. As an example, we have targeted mouse VEGF-A promoter with small hairpin RNAs (shRNAs) and identified two shRNAs which either repressed or induced VEGF-A expression on messenger RNA and protein level in vitro, depending on the targeted location. The changes in expression levels correlate with changes in the levels of epigenetic markers, such as histone modifications associated with repressed or active state of chromatin. In ischemic mouse hindlimbs, upregulation of VEGF-A expression increased vascularity and blood flow. When VEGF-A was upregulated in mouse myocardial infarction model, the blood vessel formation in the risk zone was observed and infarct size was significantly decreased already 2 weeks after treatment. We suggest that epigenetic upregulation of VEGF-A by ncRNAs can be transferred to clinical use for the treatment of ischemic diseases in the near future.

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry.

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5' end nonsense mutation (c.97G>T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin-α and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD.

Genome-wide screen of cell-cycle regulators in normal and tumor cells identifies a differential response to nucleosome depletion.

To identify cell cycle regulators that enable cancer cells to replicate DNA and divide in an unrestricted manner, we performed a parallel genome-wide RNAi screen in normal and cancer cell lines. In addition to many shared regulators, we found that tumor and normal cells are differentially sensitive to loss of the histone genes transcriptional regulator CASP8AP2. In cancer cells, loss of CASP8AP2 leads to a failure to synthesize sufficient amount of histones in the S-phase of the cell cycle, resulting in slowing of individual replication forks. Despite this, DNA replication fails to arrest, and tumor cells progress in an elongated S-phase that lasts several days, finally resulting in death of most of the affected cells. In contrast, depletion of CASP8AP2 in normal cells triggers a response that arrests viable cells in S-phase. The arrest is dependent on p53, and preceded by accumulation of markers of DNA damage, indicating that nucleosome depletion is sensed in normal cells via a DNA-damage -like response that is defective in tumor cells.

Oncogenic Herpesvirus Utilizes Stress-Induced Cell Cycle Checkpoints for Efficient Lytic Replication.

Kaposi's sarcoma herpesvirus (KSHV) causes Kaposi's sarcoma and certain lymphoproliferative malignancies. Latent infection is established in the majority of tumor cells, whereas lytic replication is reactivated in a small fraction of cells, which is important for both virus spread and disease progression. A siRNA screen for novel regulators of KSHV reactivation identified the E3 ubiquitin ligase MDM2 as a negative regulator of viral reactivation. Depletion of MDM2, a repressor of p53, favored efficient activation of the viral lytic transcription program and viral reactivation. During lytic replication cells activated a p53 response, accumulated DNA damage and arrested at G2-phase. Depletion of p21, a p53 target gene, restored cell cycle progression and thereby impaired the virus reactivation cascade delaying the onset of virus replication induced cytopathic effect. Herpesviruses are known to reactivate in response to different kinds of stress, and our study now highlights the molecular events in the stressed host cell that KSHV has evolved to utilize to ensure efficient viral lytic replication.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Uterine leiomyoma-linked MED12 mutations disrupt mediator-associated CDK activity.

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8-driven cancers.

Epigenetic upregulation of endogenous VEGF-A reduces myocardial infarct size in mice.

"Epigenetherapy" alters epigenetic status of the targeted chromatin and modifies expression of the endogenous therapeutic gene. In this study we used lentiviral in vivo delivery of small hairpin RNA (shRNA) into hearts in a murine infarction model. shRNA complementary to the promoter of vascular endothelial growth factor (VEGF-A) was able to upregulate endogenous VEGF-A expression. Histological and multiphoton microscope analysis confirmed the therapeutic effect in the transduced hearts. Magnetic resonance imaging (MRI) showed in vivo that the infarct size was significantly reduced in the treatment group 14 days after the epigenetherapy. Importantly, we show that promoter-targeted shRNA upregulates all isoforms of endogenous VEGF-A and that an intact hairpin structure is required for the shRNA activity. In conclusion, regulation of gene expression at the promoter level is a promising new treatment strategy for myocardial infarction and also potentially useful for the upregulation of other endogenous genes.

Identification of candidate oncogenes in human colorectal cancers with microsatellite instability.

Microsatellite instability can be found in approximately 15% of all colorectal cancers. To detect new oncogenes we sequenced the exomes of 25 colorectal tumors and respective healthy colon tissue. Potential mutation hot spots were confirmed in 15 genes; ADAR, DCAF12L2, GLT1D1, ITGA7, MAP1B, MRGPRX4, PSRC1, RANBP2, RPS6KL1, SNCAIP, TCEAL6, TUBB6, WBP5, VEGFB, and ZBTB2; these were validated in 86 tumors with microsatellite instability. ZBTB2, RANBP2, and PSRC1 also were found to contain hot spot mutations in the validation set. The form of ZBTB2 associated with colorectal cancer increased cell proliferation. The mutation hot spots might be used to develop personalized tumor profiling and therapy.