ASAH1 facilitates TNBC by DUSP5 suppression-driven activation of MAP kinase pathway and represents a therapeutic vulnerability.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is prone to metastasis and therapy resistance. Owing to its aggressive nature and limited availability of targeted therapies, TNBC is associated with higher mortality as compared to other forms of breast cancer. In order to develop new therapeutic options for TNBC, we characterized the factors involved in TNBC growth and progression. Here, we demonstrate that N-acylsphingosine amidohydrolase 1 (ASAH1) is overexpressed in TNBC cells and is regulated via p53 and PI3K-AKT signaling pathways. Genetic knockdown or pharmacological inhibition of ASAH1 suppresses TNBC growth and progression. Mechanistically, ASAH1 inhibition stimulates dual-specificity phosphatase 5 (DUSP5) expression, suppressing the mitogen-activated protein kinase (MAPK) pathway. Furthermore, pharmacological cotargeting of the ASAH1 and MAPK pathways inhibits TNBC growth. Collectively, we unmasked a novel role of ASAH1 in driving TNBC and identified dual targeting of the ASAH1 and MAPK pathways as a potential new therapeutic approach for TNBC treatment.

ATAD2 is a driver and a therapeutic target in ovarian cancer that functions by upregulating CENPE.

Ovarian cancer is a complex disease associated with multiple genetic and epigenetic alterations. The emergence of treatment resistance in most patients causes ovarian cancer to become incurable, and novel therapies remain necessary. We identified epigenetic regulator ATPase family AAA domain-containing 2 (ATAD2) is overexpressed in ovarian cancer and is associated with increased incidences of metastasis and recurrence. Genetic knockdown of ATAD2 or its pharmacological inhibition via ATAD2 inhibitor BAY-850 suppressed ovarian cancer growth and metastasis in both in vitro and in vivo models. Transcriptome-wide mRNA expression profiling of ovarian cancer cells treated with BAY-850 revealed that ATAD2 inhibition predominantly alters the expression of centromere regulatory genes, particularly centromere protein E (CENPE). In ovarian cancer cells, changes in CENPE expression following ATAD2 inhibition resulted in cell-cycle arrest and apoptosis induction, which led to the suppression of ovarian cancer growth. Pharmacological CENPE inhibition phenotypically recapitulated the cellular changes induced by ATAD2 inhibition, and combined pharmacological inhibition of both ATAD2 and CENPE inhibited ovarian cancer cell growth more potently than inhibition of either alone. Thus, our study identified ATAD2 as regulators of ovarian cancer growth and metastasis that can be targeted either alone or in combination with CENPE inhibitors for effective ovarian cancer therapy.

LIMK2 promotes melanoma tumor growth and metastasis through G3BP1-ESM1 pathway-mediated apoptosis inhibition.

Melanoma is the leading cause of skin cancer-related deaths, and current melanoma therapies, including targeted therapies and immunotherapies, benefit only a subset of metastatic melanoma patients due to either intrinsic or acquired resistance. LIM domain kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIMK2 is overexpressed in melanoma, and its genetic or pharmacological inhibition impairs melanoma tumor growth and metastasis in both cell culture and mice. To determine the mechanism by which LIMK2 promotes melanoma tumor growth and metastatic progression, we performed a phosphoproteomics analysis and identified G3BP1 as a key LIMK2 target, which mirrored the effects of LIMK2 inhibition when inhibited. To further determine the role of G3BP1 downstream of LIMK2, we knocked down the expression of G3BP1, performed RNA-seq analysis, and identified ESM1 as a downstream target of G3BP1. G3BP1 was required for ESM1 mRNA stability, and ESM1 ectopic expression rescued LIMK2 or G3BP1 inhibition-induced suppression of melanoma growth and metastatic attributes. These results collectively identify the LIMK2→G3BP1→ESM1 pathway as a facilitator of melanoma tumor growth and metastasis and document that LIMK2 is a therapeutically tractable target for melanoma therapy.

HOXC6 drives a therapeutically targetable pancreatic cancer growth and metastasis pathway by regulating MSK1 and PPP2R2B.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, which lacks effective therapies. Here, we demonstrate that the transcription factor, homeobox C6 (HOXC6), is overexpressed in most PDACs, and its inhibition blocks PDAC tumor growth and metastasis. HOXC6 transcriptionally activates tumor-promoting kinase MSK1 and suppresses tumor-inhibitory protein PPP2R2B in PDAC. HOXC6-induced PPP2R2B suppression causes mammalian target of rapamycin (mTOR) pathway activation, which facilitates PDAC growth. Also, MSK1 upregulation by HOXC6 is necessary for PDAC growth because of its ability to suppress apoptosis via its substrate DDX17. Combinatorial pharmacological inhibition of MSK1 and mTOR potently suppressed PDAC tumor growth and metastasis in PDAC mouse models. PDAC cells with acquired resistance to MSK1/mTOR-inhibitors displayed activated insulin-like growth factor 1 receptor (IGF1R) signaling and were successfully eradicated by IGF1R inhibitor. Furthermore, MEK inhibitor trametinib enhanced the efficacy of dual MSK1 and mTOR inhibition. Collectively, these results identify therapeutic vulnerabilities of PDAC and an approach to overcome acquired drug resistance to prolong therapeutic benefit.

Co-targeting of specific epigenetic regulators in combination with CDC7 potently inhibit melanoma growth.

Melanoma is a highly aggressive skin cancer that frequently metastasizes, but current therapies only benefit some patients. Here, we demonstrate that the serine/threonine kinase cell division cycle 7 (CDC7) is overexpressed in melanoma, and patients with higher expression have shorter survival. Transcription factor ELK1 regulates CDC7 expression, and CDC7 inhibition promotes cell cycle arrest, senescence, and apoptosis, leading to inhibition of melanoma tumor growth and metastasis. Our chemical genetics screen with epigenetic inhibitors revealed stronger melanoma tumor growth inhibition when XL413 is combined with the EZH2 inhibitor GSK343 or BRPF1/2/3 inhibitor OF1. Mechanistically, XL413 with GSK343 or OF1 synergistically altered the expression of tumor-suppressive genes, leading to higher apoptosis than the single agent alone. Collectively, these results identify CDC7 as a driver of melanoma tumor growth and metastasis that can be targeted alone or in combination with EZH2 or BRPF1/2/3 inhibitors.

Betacellulin promotes tumor development and EGFR mutant lung cancer growth by stimulating the EGFR pathway and suppressing apoptosis.

Oncogenic mutations in the EGFR gene account for 15-20% of lung adenocarcinoma (LUAD) cases. However, the mechanism for EGFR driven tumor development and growth is not fully understood. Here, using an mRNA expression profiling-based approach we identified betacellulin (BTC) as one the gene upregulated by oncogenic EGFR in an MAP kinase-dependent manner. BTC protein expression was markedly increased in LUAD patient samples compared to normal lung tissue, with higher expression in EGFR-mutant LUAD. BTC was sufficient to transform immortalized mouse cells, initiate tumor development in mice, and promote the survival of immortalized human lung epithelial cells. Conversely, knockdown of BTC inhibited the growth of EGFR-mutant human LUAD cells in culture and their tumor-forming ability in mice. Mechanistically, BTC knockdown resulted in attenuated EGFR signaling and apoptosis induction. Collectively, these results demonstrate a key role of BTC in EGFR-mutant LUAD, with potential therapeutic implications in LUAD and other EGFR-mutant cancers.

High glutamine suppresses osteogenesis through mTORC1-mediated inhibition of the mTORC2/AKT-473/RUNX2 axis.

Activation of the key nutrient cellular sensors mTORC1 and mTORC2 directs the fate of mesenchymal stromal cells (MSCs). Here, we report that glutamine regulates crosstalk between mTOR complexes and lineage commitment of MSCs independent of glucose concentration. High glutamine-induced mTORC1 hyperactivation resulted in the suppression of mTORC2, which otherwise stabilizes RUNX2 via GSK3ß inhibition through pAKT-473. Activation of GSK3ß resulted in the ubiquitination of RUNX2, a key transcription factor for the osteogenic commitment of MSCs. However, low glutamine conditions inhibit mTORC1 hyperactivation followed by increased mTORC2 activation and RUNX2 stabilization. Under diabetic/high-glucose conditions, glutamine-triggered hyperactivation of mTORC1 resulted in mTORC2 suppression, and active GSK3ß led to suppression of RUNX2. Activation of p-AMPK by metformin inhibits high glutamine-induced mTORC1 hyperactivation and rescues RUNX2 through the mTORC2/AKT-473 axis. Collectively, our study indicates the role of glutamine in modulating MSC fate through cross-talk between mTOR complexes by identifying a critical switch in signaling. It also shows the importance of glutamine in modulating molecular cues (mTORC1/p-70S6K/mTORC2/RUNX2) that are involved in driving diabetes-induced bone adipogenesis and other secondary complications.

Disruptor of telomeric silencing 1-like promotes ovarian cancer tumor growth by stimulating pro-tumorigenic metabolic pathways and blocking apoptosis.

Ovarian cancer is the leading cause of gynecological malignancy-related deaths. Current therapies for ovarian cancer do not provide meaningful and sustainable clinical benefits, highlighting the need for new therapies. We show that the histone H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) is overexpressed in ovarian cancer and that a higher level of DOT1L expression correlates with shorter progression-free and overall survival (OS). Pharmacological inhibition of DOT1L (EPZ-5676, EPZ004777, and SGC0946) or genetic inhibition of DOT1L attenuates the growth of ovarian cancer cells in cell culture and in a mouse xenograft model of ovarian cancer. Transcriptome-wide mRNA expression profiling shows that DOT1L inhibition results in the downregulation of genes involved in cellular biosynthesis pathways and the upregulation of proapoptotic genes. Consistent with the results of transcriptome analysis, the unbiased large-scale metabolomic analysis showed reduced levels of several metabolites of the amino acid and nucleotide biosynthesis pathways after DOT1L inhibition. DOT1L inhibition also resulted in the upregulation of the NKG2D ligand ULBP1 and subsequent increase in natural killer (NK) cell-mediated ovarian cancer eradication. Collectively, our results demonstrate that DOT1L promotes ovarian cancer tumor growth by regulating apoptotic and metabolic pathways as well as NK cell-mediated eradication of ovarian cancer and identifies DOT1L as a new pharmacological target for ovarian cancer therapy.

The BRD9/7 Inhibitor TP-472 Blocks Melanoma Tumor Growth by Suppressing ECM-Mediated Oncogenic Signaling and Inducing Apoptosis.

Melanoma accounts for the majority of all skin cancer-related deaths and only 1/3rd of melanoma patients with distal metastasis survive beyond five years. However, current therapies including BRAF/MEK targeted therapies or immunotherapies only benefit a subset of melanoma patients due to the emergence of intrinsic or extrinsic resistance mechanisms. Effective treatment of melanoma will thus require new and more effective therapeutic agents. Towards the goal of identifying new therapeutic agents, we conducted an unbiased, druggable epigenetic drug screen using a library of 32 epigenetic inhibitors obtained from the Structural Genome Consortium that targets proteins encoding for epigenetic regulators. This chemical genetic screening identified TP-472, which targets bromodomain-7/9, as the strongest inhibitor of melanoma growth in both short- and long-term survival assays and in mouse models of melanoma tumor growth. Mechanistically, using a transcriptome-wide mRNA sequencing profile we identified TP-472 treatment downregulates genes encoding various extracellular matrix (ECM) proteins, including integrins, collagens, and fibronectins. Reactome-based functional pathway analyses revealed that many of the ECM proteins are involved in extracellular matrix interactions required for cancer cell growth and proliferation. TP-472 treatment also upregulated several pro-apoptotic genes that can inhibit melanoma growth. Collectively, our results identify BRD7/9 inhibitor TP-472 as a potentially useful therapeutic agent for melanoma therapy.

Identification of the Mutational Landscape of Gynecological Malignancies.

Background: Cancer is a complex disease that arises from the accumulation of multiple genetic and non-genetic changes. Advances in sequencing technologies have allowed unbiased and global analysis of patient-derived tumor samples and the discovery of genetic and transcriptional changes in key genes and oncogenic pathways. That in turn has facilitated a better understanding of the underlying causes of cancer initiation and progression, resulting in new therapeutic targets. Methods: In our study, we have analyzed the mutational landscape of gynecological malignancies using datasets from The Cancer Genome Atlas (TCGA). We have also analyzed Oncomine datasets to establish the impact of their alteration on disease recurrence and survival of patients. Results: In this study, we analyzed a series of different gynecological malignancies for commonly occurring genetic and non-genetic alterations. These studies show that white women have higher incidence of gynecological malignancies. Furthermore, our study identified 16 genes that are altered at a frequency >10% among all of the gynecological malignancies and tumor suppressor TP53 is the most altered gene in these malignancies (>50% of the cases). The top 16 genes fall into the categories of either tumor suppressor or oncogenes and a subset of these genes are associated with poor prognosis, some affecting recurrence and survival of ovarian cancer patients. Conclusion: In sum, our study identified 16 major genes that are broadly mutated in a large majority of gynecological malignancies and in some cases predict survival and recurrence in patients with gynecological malignancies. We predict that the functional studies will determine their relative role in the initiation and progression of gynecological malignancies and also establish if some of them represents drug targets for anti-cancer therapy.

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells' ability to eradicate dysfunctional target cells.

LIMK2 promotes the metastatic progression of triple-negative breast cancer by activating SRPK1.

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer subtype and due to the lack of hormone receptors and HER2 expression, TNBC has limited therapeutic options with chemotherapy being the primary choice for systemic therapy. LIM Domain Kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIM domain kinase 2 (LIMK2) is overexpressed in TNBC, and short-hairpin RNA (shRNA)-mediated LIMK2 knockdown or its pharmacological inhibition blocks metastatic attributes of TNBC cells. To determine the mechanism by which LIMK2 promotes TNBC metastatic progression, we performed stable isotope labeling by amino acids in cell culture (SILAC) based unbiased large-scale phosphoproteomics analysis. This analysis identified 258 proteins whose phosphorylation was significantly reduced due to LIMK2 inhibition. Among these proteins, we identified SRSF protein kinase 1 (SRPK1), which encodes for a serine/arginine protein kinase specific for the SR (serine/arginine-rich domain) family of splicing factors. We show that LIMK2 inhibition blocked SRPK1 phosphorylation and consequentially its activity. Furthermore, similar to LIMK2, genetic inhibition of SRPK1 by shRNAs or its pharmacological inhibition blocked the metastatic attributes of TNBC cells. Moreover, the pharmacological inhibition of LIMK2 blocked metastatic progression in mice without affecting primary tumor growth. In sum, these results identified LIMK2 as a facilitator of distal TNBC metastasis and a potential target for preventing TNBC metastatic progression.

Loss of HAT1 expression confers BRAFV600E inhibitor resistance to melanoma cells by activating MAPK signaling via IGF1R.

BRAF inhibitors (BRAFi) have been approved for the clinical treatment of BRAF-mutant metastatic melanoma. Although initial responses to BRAFi are generally favorable, acquired BRAFi resistance emerges rapidly, resulting in treatment failure. Only some of the underlying mechanisms responsible for BRAFi resistance are currently understood. Here, we showed that the genetic inhibition of histone acetyltransferase 1 (HAT1) in BRAF-mutant melanoma cells resulted in BRAFi resistance. Using quantitative immunofluorescence analysis of patient sample pairs, consisting of pre-treatment along with matched progressed BRAFi + MEKi-treated melanoma samples, HAT1 downregulation was observed in 7/11 progressed samples (~63%) in comparison with pre-treated samples. Employing NanoString-based nCounter PanCancer Pathway Panel-based gene expression analysis, we identified increased MAPK, Ras, transforming growth factor (TGF)-ß, and Wnt pathway activation in HAT1 expression inhibited cells. We further found that MAPK pathway activation following the loss of HAT1 expression was partially driven by increased insulin growth factor 1 receptor (IGF1R) signaling. We showed that both MAPK and IGF1R pathway inhibition, using the ERK inhibitor SCH772984 and the IGF1R inhibitor BMS-754807, respectively, restored BRAFi sensitivity in melanoma cells lacking HAT1. Collectively, we show that the loss of HAT1 expression confers acquired BRAFi resistance by activating the MAPK signaling pathway via IGF1R.

A novel phosphorylation by AMP-activated kinase regulates RUNX2 from ubiquitination in osteogenesis over adipogenesis.

Mesenchymal stem cells (MSCs) function as progenitors to a variety of cell types. The reported association between osteogenic and adipogenic commitment during differentiation is due to the regulation of key transcription factors in the signaling pathways. However, the process of adipogenesis at the expense of osteogenic phenotype during metabolic stress is still unclear. In this study, we showed for the first time that RUNX2 is a novel substrate of AMP-activated kinase (AMPK), which directly phosphorylates at serine 118 residue in the DNA-binding domain of RUNX2. Our results in in vitro MSC lineage differentiation models confirmed that active AMPK and RUNX2-S118 phosphorylation are preferentially associated with osteogenic commitment, whereas the lack of this phosphorylation leads to adipogenesis. This interplay is regulated by the ubiquitination of non-phosphorylated RUNX2-S118, which is evident in the dominant mutant RUNX2-S118D. Pharmacological activation of AMPK by metformin significantly abrogated the loss of RUNX2-S118 phosphorylation and protected from tunicamycin-induced endoplasmic reticulum stress, high glucose-induced in vitro adipogenesis and streptozotocin-induced in vivo bone adiposity and bone phenotype. In conclusion, results from this study demonstrated that RUNX2 is a direct target of AMPK which simplified the outlook towards several complex mechanisms that are currently established concerning cellular metabolism and pathogenesis.