ALK inhibitors suppress HCC and synergize with anti-PD-1 therapy and ABT-263 in preclinical models.

Hepatocellular carcinoma (HCC) currently lacks effective therapies, leaving a critical need for new treatment options. A previous study identified the anaplastic lymphoma kinase (ALK) amplification in HCC patients, raising the question of whether ALK inhibitors could be a viable treatment. Here, we showed that both ALK inhibitors and ALK knockout effectively halted HCC growth in cell cultures. Lorlatinib, a potent ALK inhibitor, suppressed HCC tumor growth and metastasis across various mouse models. Additionally, in an advanced immunocompetent humanized mouse model, when combined with an anti-PD-1 antibody, lorlatinib more potently suppressed HCC tumor growth, surpassing individual drug efficacy. Lorlatinib induced apoptosis and senescence in HCC cells, and the senolytic agent ABT-263 enhanced the efficacy of lorlatinib. Additional studies identified that the apoptosis-inducing effect of lorlatinib was mediated via GGN and NRG4. These findings establish ALK inhibitors as promising HCC treatments, either alone or in combination with immunotherapies or senolytic agents.

Roles and therapeutic targeting of ceramide metabolism in cancer.

BACKGROUND: Ceramides are sphingolipids that act as signaling molecules involved in regulating cellular processes including apoptosis, proliferation, and metabolism. Deregulation of ceramide metabolism contributes to cancer development and progression. Therefore, regulation of ceramide levels in cancer cells is being explored as a new approach for cancer therapy. SCOPE OF THE REVIEW: This review discusses the multiple roles of ceramides in cancer cells and strategies to modulate ceramide levels for cancer therapy. Ceramides attenuate cell survival signaling and metabolic pathways, while activating apoptotic mechanisms, making them tumor-suppressive. Approaches to increase ceramide levels in cancer cells include using synthetic analogs, inhibiting ceramide degradation, and activating ceramide synthesis. We also highlight combination therapies such as use of ceramide modulators with chemotherapies, immunotherapies, apoptosis inducers, and anti-angiogenics, which offer synergistic antitumor effects. Additionally, we also describe ongoing clinical trials evaluating ceramide nanoliposomes and analogs. Finally, we discuss the challenges of these therapeutic approaches including the complexity of ceramide metabolism, targeted delivery, cancer heterogeneity, resistance mechanisms, and long-term safety. MAJOR CONCLUSIONS: Ceramide-based therapy is a potentially promising approach for cancer therapy. However, overcoming hurdles in pharmacokinetics, specificity, and resistance is needed to optimize its efficacy and safety. This requires comprehensive preclinical/clinical studies into ceramide signaling, formulations, and combination therapies. Ceramide modulation offers opportunities for developing novel cancer treatments, but a deeper understanding of ceramide biology is vital to advance its clinical applications.

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.

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.

CBX5 loss drives EGFR inhibitor resistance and results in therapeutically actionable vulnerabilities in lung cancer.

Although epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFRi) are approved for treating EGFR-mutant lung adenocarcinoma (LUAD), emergence of acquired resistance limits their clinical benefits. Several mechanisms for acquired resistance to EGFRi in LUAD have been identified; however, the molecular basis for this resistance remains unknown in ~30% of LUAD. Chromatin and DNA modifiers and their regulators play important roles in determining response to anticancer therapies. Therefore, to identify nongenetic mechanisms of EGFRi resistance in LUAD, we performed an epigenome-wide shRNA screen targeting 363 human epigenetic regulator genes. This screen identified loss of the transcriptional repressor chromobox homolog 5 (CBX5) as a driver of EGFRi resistance in EGFR-mutant LUAD. Loss of CBX5 confers resistance to multiple EGFRi in both cell culture and mice. We found that CBX5 loss in EGFR-mutant LUAD cells leads to increased expression of the transcription factor E2F1, which in turn stimulates expression of the antiapoptotic gene BIRC5 (survivin). This E2F1-mediated upregulation of BIRC5 in CBX5-knockdown LUAD cells attenuates apoptosis induction following EGFRi treatment. Consistent with these results, knockdown of E2F1 or BIRC5 partly rescues CBX5-knockdown-induced EGFRi resistance in cell culture and mice. EGFRi-resistant LUAD cell lines show reduced CBX5 expression compared to parental lines; however, bromo- and extra-terminal (BET)-domain inhibitors (BETi) restore CBX5 expression in these cells and sensitize them to EGFRi/BETi combination therapy. Similarly, treatment with a BIRC5 inhibitor suppresses growth of EGFRi-resistant LUAD cells. Collectively, these studies identify CBX5 loss as a driver of EGFRi resistance and reveal therapeutic opportunities for treating EGFRi-resistant LUAD.

UBE2T promotes breast cancer tumor growth by suppressing DNA replication stress.

Breast cancer is a leading cause of cancer-related deaths among women, and current therapies benefit only a subset of these patients. Here, we show that ubiquitin-conjugating enzyme E2T (UBE2T) is overexpressed in patient-derived breast cancer samples, and UBE2T overexpression predicts poor prognosis. We demonstrate that the transcription factor AP-2 alpha (TFAP2A) is necessary for the overexpression of UBE2T in breast cancer cells, and UBE2T inhibition suppresses breast cancer tumor growth in cell culture and in mice. RNA sequencing analysis identified interferon alpha-inducible protein 6 (IFI6) as a key downstream mediator of UBE2T function in breast cancer cells. Consistently, UBE2T inhibition downregulated IFI6 expression, promoting DNA replication stress, cell cycle arrest, and apoptosis and suppressing breast cancer cell growth. Breast cancer cells with IFI6 inhibition displayed similar phenotypes as those with UBE2T inhibition, and ectopic IFI6 expression in UBE2T-knockdown breast cancer cells prevented DNA replication stress and apoptosis and partly restored breast cancer cell growth. Furthermore, UBE2T inhibition enhanced the growth-suppressive effects of DNA replication stress inducers. Taken together, our study identifies UBE2T as a facilitator of breast cancer tumor growth and provide a rationale for targeting UBE2T for breast cancer therapies.

Transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor-resistant pancreatic ductal adenocarcinoma.

Metabolic reprogramming, due in part to the overexpression of metabolic enzymes, is a key hallmark of cancer cells. Lactate dehydrogenase (LDHA), a metabolic enzyme that catalyzes the interconversion of lactate and pyruvate, is overexpressed in a wide variety of cancer types, including pancreatic ductal adenocarcinoma (PDAC). Furthermore, the genetic or pharmacological inhibition of LDHA suppresses cancer growth, demonstrating a cancer-promoting role for this enzyme. Therefore, several pharmacological LDHA inhibitors are being developed and tested as potential anti-cancer therapeutic agents. Because cancer cells are known to rapidly adapt and become resistant to anti-cancer therapies, in this study, we modeled the adaptation of cancer cells to LDHA inhibition. Using PDAC as a model system, we studied the molecular aspects of cells resistant to the competitive LDHA inhibitor sodium oxamate. We performed unbiased RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), and metabolomics analyses of parental and oxamate-resistant PDAC cells treated with and without oxamate to identify the transcriptional, chromatin, and metabolic landscapes of these cells. We found that oxamate-resistant PDAC cells were significantly different from parental cells at the levels of mRNA expression, chromatin accessibility, and metabolites. Additionally, an integrative analysis combining the RNA-seq and ATAC-seq datasets identified a subset of differentially expressed mRNAs that directly correlated with changes in chromatin accessibility. Finally, functional analysis of differentially expressed metabolic genes in parental and oxamate-resistant PDAC cells treated with and without oxamate, together with an integrative analysis of RNA-seq and metabolomics data, revealed changes in metabolic enzymes that might explain the changes in metabolite levels observed in these cells. Collectively, these studies identify the transcriptional, chromatin, and metabolic landscapes of LDHA inhibitor resistance in PDAC cells. Future functional studies related to these changes remain necessary to reveal the direct roles played by these changes in the development of LDHA inhibitor resistance and uncover approaches for more effective use of LDHA inhibitors in cancer therapy.

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.

Changes in the Transcriptome and Chromatin Landscape in BRAFi-Resistant Melanoma Cells.

Metastatic and drug-resistant melanoma are leading causes of skin cancer-associated death. Mitogen-associated protein kinase (MAPK) pathway inhibitors and immunotherapies have provided substantial benefits to patients with melanoma. However, long-term therapeutic efficacy has been limited due to emergence of treatment resistance. Despite the identification of several molecular mechanisms underlying the development of resistant phenotypes, significant progress has still not been made toward the effective treatment of drug-resistant melanoma. Therefore, the identification of new targets and mechanisms driving drug resistance in melanoma represents an unmet medical need. In this study, we performed unbiased RNA-sequencing (RNA-seq) and assay for transposase-accessible chromatin with sequencing (ATAC-seq) to identify new targets and mechanisms that drive resistance to MAPK pathway inhibitors targeting BRAF and MAPK kinase (MEK) in BRAF-mutant melanoma cells. An integrative analysis of ATAC-seq combined with RNA-seq showed that global changes in chromatin accessibility affected the mRNA expression levels of several known and novel genes, which consequently modulated multiple oncogenic signaling pathways to promote resistance to MAPK pathway inhibitors in melanoma cells. Many of these genes were also associated with prognosis predictions in melanoma patients. This study resulted in the identification of new genes and signaling pathways that might be targeted to treat MEK or BRAF inhibitors resistant melanoma patients. The present study applied new and advanced approaches to identify unique changes in chromatin accessibility regions that modulate gene expression associated with pathways to promote the development of resistance to MAPK pathway 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.

Epigenetic regulation and targeting of ECM for cancer therapy.

The tumor microenvironment (TME) composed of different types of cells embedded in extracellular matrix (ECM) has crucial effects on cancer growth and metastasis. ECM is made of a variety of proteins that provide structural support to the cells and regulate biological functions by modulating the cross talk among cells, thus effecting tumor growth and progression. In this mini-review, the author discusses epigenetic modifications that regulate the expression of fibrous ECM proteins and glycoproteins and the prospects of targeting them for cancer therapy.

Transcriptional determinants of cancer immunotherapy response and resistance.

The host immune response is a potent defense mechanism against cancer development and progression. To survive, cancer cells must develop mechanisms to evade the immune response. Based on this knowledge, a series of new therapies collectively referred to as immunotherapies have been developed and translated to the clinic for treating cancer patients. Although some cancer subtypes have shown strong clinical responses, including curative outcomes in some patients, immunotherapies have not worked as desired for some subtypes and forms of cancers. We provide an overview of the transcriptional mechanisms that drive the response and resistance to immunotherapies. We also discuss possible interventions to enhance the outcomes of immunotherapies by targeting dysregulated transcriptional networks in cancer cells.

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.

Epigenetic Alterations and Mechanisms That Drive Resistance to Targeted Cancer Therapies.

Cancer is a complex disease and cancer cells typically harbor multiple genetic and epigenetic alterations. Large-scale sequencing of patient-derived cancer samples has identified several druggable driver oncogenes. Many of these oncogenes can be pharmacologically targeted to provide effective therapies for breast cancer, leukemia, lung cancer, melanoma, lymphoma, and other cancer types. Initial responses to these agents can be robust in many cancer types and some patients with cancer experience sustained tumor inhibition. However, resistance to these targeted therapeutics frequently emerges, either from intrinsic or acquired mechanisms, posing a major clinical hurdle for effective treatment. Several resistance mechanisms, both cell autonomous and cell nonautonomous, have been identified in different cancer types. Here we describe how alterations of the transcriptome, transcription factors, DNA, and chromatin regulatory proteins confer resistance to targeted therapeutic agents. We also elaborate on how these studies have identified underlying epigenetic factors that drive drug resistance and oncogenic pathways, with direct implications for the prevention and treatment of drug-resistant cancer.

EZH2 inhibits NK cell-mediated antitumor immunity by suppressing CXCL10 expression in an HDAC10-dependent manner.

Enhancer of zeste homolog 2 (EZH2) is a histone H3 lysine 27 methyltransferase that has been shown to function as an oncogene in some cancers. Previous reports have largely focused on the ability of EZH2 to regulate cell-intrinsic tumor regulatory pathways as its mechanism-of-oncogenic action. However, the role that EZH2-mediated immune suppression plays in its oncogenic activity is not fully known. In particular, the role of natural killer (NK) cells in EZH2-driven tumor growth remains incompletely understood. Here, we demonstrate that genetic or pharmacological inhibition of EZH2 induces reexpression of the chemokine CXCL10 in hepatic tumor cells. We find that histone deacetylase 10 (HDAC10) is necessary for EZH2 recruitment to the CXCL10 promoter, leading to CXCL10 transcriptional repression. Critically, CXCL10 is necessary and sufficient for stimulating NK cell migration, and EZH2's ability to inhibit NK cell migration via CXCL10 suppression is conserved in other EZH2-dependent cancers. NK cell depletion in an immunocompetent syngeneic mouse model of hepatic tumorigenesis reverses the tumor inhibitory effects of an EZH2 inhibitor (GSK343), and inhibitor-mediated reexpression of CXCL10 is required for its tumor suppressive effects in the same mouse model. Collectively, these results reveal a decisive role for NK cells and CXCL10 in mediating the oncogenic function of EZH2.

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.

PSPH promotes melanoma growth and metastasis by metabolic deregulation-mediated transcriptional activation of NR4A1.

Metabolic deregulation, a hallmark of cancer, fuels cancer cell growth and metastasis. Here, we show that phosphoserine phosphatase (PSPH), an enzyme of the serine metabolism pathway, is upregulated in patient-derived melanoma samples. PSPH knockdown using short hairpin RNAs (shRNAs) blocks melanoma tumor growth and metastasis in both cell culture and mice. To elucidate the mechanism underlying PSPH action, we evaluated PSPH shRNA-expressing melanoma cells using global metabolomics and targeted mRNA expression profiling. Metabolomics analysis showed an increase in 2-hydroxyglutarate (2-HG) levels in PSPH knockdown cells. 2-HG inhibits the TET family of DNA demethylases and the Jumonji family of histone demethylases (KDM and JMJD), which is known to impact gene expression. Consistent with these data, PSPH knockdown in melanoma cells showed reduced DNA 5-hydroxymethylcytosine (5hmC) and increased histone H3K4me3 modifications. 2-HG treatment also inhibited melanoma growth. The nCounter PanCancer Pathways Panel-based mRNA expression profiling revealed attenuation of a number of cancer-promoting pathways upon PSPH knockdown. In particular, PSPH was necessary for nuclear receptor NR4A1 expression. Ectopic NR4A1 expression partly rescued the growth of melanoma cells expressing PSPH shRNA. Collectively, these results link PSPH to the facilitation of melanoma growth and metastasis through suppression of 2-HG and thus activation of pro-oncogenic gene expression.

Transcriptional regulators and alterations that drive melanoma initiation and progression.

Although melanoma is the least frequent type of skin cancer, it accounts for the majority of skin cancer-related deaths. Large-scale sequencing efforts have led to the classification of melanoma into four major subtypes (i.e., BRAF-mutant, NRAS-mutant, NF1-deficient, and triple wild-type). These sequencing studies have also revealed that melanoma genomes are some of the most mutated genomes of all cancers and therefore have a high neoantigen load. These findings have resulted in the development and clinical use of targeted therapies against the oncogenic BRAF→MEK→ERK pathway and immune checkpoint inhibitors for the treatment of metastatic melanoma. Although some patients with metastatic melanoma benefit immensely from these transformative therapies, others either become resistant or do not respond at all. These clinical challenges have intensified the search for new drug targets and drugs that can benefit patients who are either intrinsically resistant or have acquired resistance to targeted therapies and immunotherapies. Numerous signaling pathways and oncogenic drivers can cause changes in mRNA transcription that in turn drive melanoma initiation and progression. Transcriptional regulation of mRNA expression is necessary to maintain cell identity and cellular plasticity via the regulation of transcription factor expression and function, promoter/enhancer activities, chromatin regulators, and three-dimensional genome organization. Transcriptional deregulation can arise due to genetic and/or non-genetic alterations in the genome. Specifically, these deregulated transcriptional programs can become liabilities for melanoma cells due to their acquired dependencies on these programs for survival, which can be harnessed to develop new therapies for melanoma. In this article, we present an overview of the mechanisms that result in the transcriptional deregulation of mRNA expression in melanoma cells and assess how these changes facilitate melanoma initiation and progression. We also describe how these deregulated transcriptional pathways represent new opportunities for the development of unconventional and potentially impactful treatments for metastatic melanoma.

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.

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.