The SRY-HMG-Box transcription factor SOX10 plays a critical role in neural crest development, but its function in epithelial tumorigenesis remains unclear. Here, we identify SOX10 as a key regulator of tumor-initiating activity in Neu-driven mammary cancers. Genetic ablation of Sox10 in the luminal compartment of MMTV-Neu (NIC) mice resulted in delayed but normal mammary gland development. Sox10 deletion resulted in a reduction in mammary progenitors and a complete loss of tumor initiation in Sox10-deficient luminal cells. CRISPR/Cas9-mediated Sox10 inactivation in Neu-transformed tumor cells led to diminished self-renewal in mammosphere assays, markedly impaired growth in orthotopic transplant models and profoundly reduced lung colonization following tail vein injection, suggesting a depletion of cancer stem cell activity. Transcriptomic profiling revealed that Sox10-deficiency in Neu+ tumor cells induces a luminal-to-basal/mesenchymal-like shift and the downregulation of several genes associated with genetic networks regulating stemness. Collectively, these findings demonstrate that Sox10 is required for a permissive luminal cell state for Neu-driven tumor initiation and that it is critical for cancer stem cell activity and the establishment of metastases.

In melanoma, SOX9 and SOX10 are markers of the mesenchymal and melanocytic state, respectively. Using a panel of BRAFV600E positive YUMM lines, we find that, following chronic vemurafenib treatment, SOX10 is lost whereas SOX9 is induced. Overexpression or knock-down of either SOX9 or SOX10 had no impact on vemurafenib sensitivity. However, we find that SOX9 is necessary to program a vemurafenib-resistance memory state following a drug holiday in vitro. RNA-Seq studies show that the loss of Sox10 represents an intermediate state that is accompanied by the loss of Sox6 and the induction of Sox7, Sox9 and other phenotype switching markers. However, SOX7 expression is not sufficient to induce vemurafenib resistance. Upon acquired drug resistance, we observed differential chromatin accessibility in the Sox9 and Sox10 upstream regions, supporting their activation and repression, respectively. Overall, our data show that the loss of SOX10 and SOX9 induction are critical to program drug resistance. Furthermore, we show that the YUMM cell lines represent a good murine model to investigate transitions to an acquired drug resistant state.

The SRY-HMG-Box transcription factor SOX10 plays a critical role in neural crest development, but its function in epithelial tumorigenesis remains unclear. Here, we identify SOX10 as a key regulator of tumor-initiating activity in Neu-driven mammary cancers. Genetic ablation of Sox10 in the luminal compartment of MMTV-Neu (NIC) mice resulted in delayed but normal mammary gland development. Sox10 deletion conferred a dose-dependent delay in tumor onset, with a complete loss of tumor initiation in Sox10-deficient luminal cells. CRISPR/Cas9-mediated Sox10 inactivation in Neu-transformed tumor cells led to reduced 3D invasion and diminished self-renewal in mammosphere assays. Established Sox10-null cell lines exhibited markedly impaired growth in orthotopic transplant models and failed to colonize lung tissue following tail vein injection, suggesting a loss of tumor-initiating capacity. Transcriptomic profiling revealed that Sox10-deficiency in Neu+ tumor cells induces a luminal-to-basal/stem-like shift and the downregulation of several genes associated with genetic networks regulating stemness. Collectively, these findings demonstrate that Sox10 is required for a permissive cell progenitor state for Neu-driven tumor initiation and that it is critical to sustain the invasive and self-renewing traits that drive tumor progression and metastasis. Significance The Sox10 transcription factor is critical for mammary stem cell function and plasticity. Animal studies suggest that different subtypes of breast cancers arise from luminal epithelial cells and hypothesized the implication of luminal stem cells in breast cancer initiation. We show that Sox10 deletion in the luminal compartment of adult female mice abrogates tumor initiation. Sox10 inactivation impairs tumor cell growth, dissemination and reprograms luminal Neu+ tumor cells to a basal phenotype. Our data supports a model whereby luminal progenitors are required for tumor initiation, self-renewal and growth at distant sites. The findings provide a new therapeutic opportunity in the targeting of Sox10 networks to reduce the cancer stem cell content of mammary tumors. ### Competing Interest Statement The authors have declared no competing interest. Canadian Institute of Health Research, Ottawa,CA Cancer Research Society, https://ror.org/00t38a349 Breast Cancer Canada, Toronto, CA

Kinases are known to have kinase activity independent functions. To gain further insights into potential kinase-independent functions of SLK/STK2, we have developed a kinase-dead allele, SLKK63R using in vivo CRISPR/Cas technology. Our studies show that blastocysts homozygote for SLKK63R do not develop into viable mice. However, heterozygotes are viable and fertile with no overt phenotypes. Analyses of mouse embryonic fibroblasts show that expression of SLKK63R results in a 50% decrease in kinase activity in heterozygotes. In contrast to previous studies, our data show that SLK does not form homodimers and that the kinase defective allele does not act in a dominant negative fashion. Expression of SLKK63R leads to altered Rac1 and RhoA activity, increased stress fiber formation and delayed focal adhesion turnover. Our data support a previously observed role for SLK in cell migration and suggest that at least 50% kinase activity is sufficient for embryonic development.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Targeted therapy resistance frequently develops in melanoma due to intratumor heterogeneity and epigenetic reprogramming. This also typically induces cross-resistance to immunotherapies. Whether this includes additional modes of therapy has not been fully assessed. We show that co-treatments of MAPKi with VSV-based oncolytics do not function in a synergistic fashion; rather, the MAPKis block infection. Melanoma resistance to vemurafenib further perturbs the cells’ ability to be infected by oncolytic viruses. Resistance to vemurafenib can be induced by the loss of SOX10, a common proliferative marker in melanoma. The loss of SOX10 promotes a cross-resistant state by further inhibiting viral infection and replication. Analysis of RNA-seq datasets revealed an upregulation of interferon-stimulated genes (ISGs) in SOX10 knockout populations and targeted therapy-resistant cells. Interestingly, the induction of ISGs appears to be independent of type I IFN production. Overall, our data suggest that the pathway mediating oncolytic resistance is due to the loss of SOX10 during acquired drug resistance in melanoma.

SOX10 is a key regulator of melanoma progression and promotes a melanocytic/differentiated state. Here we identified melanoma cell lines lacking SOX10 expression which retain their in vivo growth capabilities. More importantly, we find that SOX10 can regulate T-cell infiltration in melanoma while also decreasing common cancer stem cell (CSC) properties. We show that SOX10 regulates CEACAM1, a surface protein with immunomodulatory properties. SOX10 directly binds to a distal CEACAM1 promoter region approximately 3-4kbps from the CEACAM1 transcriptional start site. Furthermore, we show that a SOX10-CEACAM1 axis can suppress CD8(+) T-cell infiltration as well as reduce CSC pool within tumors, leading to reduced tumor growth. Overall, these results identify SOX10 as a direct regulator of CEACAM1, and uncover both a pro- and anti-tumorigenic roles for SOX10 in melanoma.

The antimicrobial medication malarone (atovaquone/proguanil) is used as a fixed-dose combination for treating children and adults with uncomplicated malaria or as chemoprophylaxis for preventing malaria in travelers. It is an inexpensive, efficacious, and safe drug frequently prescribed around the world. Following anecdotal evidence from 17 patients in the provinces of Quebec and Ontario, Canada, suggesting that malarone/ atovaquone may present some benefits in protecting against COVID-19, we sought to examine its antiviral potential in limiting the replication of SARSCoV-2 in cellular models of infection. In VeroE6 expressing human TMPRSS2 and human lung Calu-3 epithelial cells, we show that the active compound atovaquone at micromolar concentrations potently inhibits the replication of SARS-CoV-2 and other variants of concern including the alpha, beta, and delta variants. Importantly, atovaquone retained its full antiviral activity in a primary human airway epithelium cell culture model. Mechanistically, we demonstrate that the atovaquone antiviral activity against SARS-CoV-2 is partially dependent on the expression of TMPRSS2 and that the drug can disrupt the interaction of the spike protein with the viral receptor, ACE2. Additionally, spike-mediated membrane fusion was also reduced in the presence of atovaquone. In the United States, two clinical trials of atovaquone administered alone or in combination with azithromycin were initiated in 2020. While we await the results of these trials, our findings in cellular infection models demonstrate that atovaquone is a potent antiviral FDA-approved drug against SARS-CoV-2 and other variants of concern in vitro.

Over the past 20 years, the Ste20-like kinase (SLK; also known as STK2) has emerged as a central regulator of cytoskeletal dynamics. Reorganization of the cytoskeleton is necessary for a plethora of biological processes including apoptosis, proliferation, migration, tissue repair and signaling. Several studies have also uncovered a role for SLK in disease progression and cancer. Here, we review the recent findings in the SLK field and summarize the various roles of SLK in different animal models and discuss the biochemical mechanisms regulating SLK activity. Together, these studies have revealed multiple roles for SLK in coupling cytoskeletal dynamics to cell growth, in muscle repair and in negative-feedback loops critical for cancer progression. Furthermore, the ability of SLK to regulate some systems appears to be kinase activity independent, suggesting that it may be an important scaffold for signal transduction pathways. These various findings reveal highly complex functions and regulation patterns of SLK in development and disease, making it a potential therapeutic target.

HER2 is overexpressed in 20–30% of all breast cancers and is associated with an invasive disease and poor clinical outcome. The Ste20-like kinase (SLK) is activated downstream of HER2/Neu and is required for efficient epithelial-to-mesenchymal transition, cell cycle progression, and migration in the mammary epithelium. Here we show that loss of SLK in a murine model of HER2/Neu-positive breast cancers significantly accelerates tumor onset and decreases overall survival. Transcriptional profiling of SLK knockout HER2/Neu-derived tumor cells revealed a strong induction in the triple-negative breast cancer marker, Sox10 , accompanied by an increase in mammary stem/progenitor activity. Similarly, we demonstrate that SLK and Sox10 expression are inversely correlated in patient samples, with the loss of SLK and acquisition of Sox10 marking the triple-negative subtype. Furthermore, pharmacological inhibition of AKT reduces SLK-null tumor growth in vivo and is rescued by ectopic Sox10 expression, suggesting that Sox10 is a critical regulator of tumor growth downstream of SLK/AKT. These findings highlight a role for SLK in negatively regulating HER2-induced mammary tumorigenesis and provide mechanistic insight into the regulation of Sox10 expression in breast cancer.

Abstract Over the past two decades, the mammalian Ste20-like kinase (SLK) has been characterized for its role in regulating cellular migration, proliferation and apoptosis in fibroblasts and myoblasts. In mammary epithelial cells, SLK has been shown to be required for efficient epithelial-to-mesenchymal transition and to be activated downstream of the HER2/Neu-oncogene to control chemotactic cellular migration. Here, we assessed the role of SLK in HER2/Neu-induced mammary tumorigenesis in vivo. As SLK is activated downstream of HER2/Neu, we hypothesized that the loss of SLK would significantly delay tumor progression in a mouse model of HER2-positive breast cancer. As we have shown that global attenuation of SLK kinase activity results in embryonic lethality, a conditional SLK knockout mouse model was generated. To study the role of SLK in HER2-positive breast cancer, we crossed these conditional SLK knockout mice with mice expressing HER2/Neu linked to Cre recombinase in the mammary luminal epithelium. Unexpectedly, we have demonstrated that conditional deletion of SLK significantly accelerates Neu-induced mammary tumor onset and decreases overall survival. SLK deletion results in the induction of Sox10 which drives mammary stem/progenitor activity and cooperates with HER2/Neu to drive tumor growth. Using the Cancer Genome Atlas, we have supported previous findings and validated Sox10 as a potential biomarker of the Triple-negative Breast Cancer subtype. Furthermore, we have uncovered that SLK deletion results in enhanced activation of AKT. We provide evidence that Sox10 induction requires signaling through a novel AKT/Sox9-dependent pathway following SLK deletion. Taken together, our data suggests that SLK may be required to maintain cells in a fully differentiated state and that loss of SLK in HER2/Neu-induced breast cancer drives a more basal/stem-like phenotype through the induction of Sox10. Citation Format: Khalid N Al-Zahrani, John Abou-Hamad, Ben Pryce, David P Cook, Johnathan Hodgins, Cedrik Labreche, Pascale Robineau-Charette, Christiano de Souza, John Bell, Rebecca Auer, Michele Ardolino, Barbara Vanderhyden, Daniel Schramek, Luc Sabourin. Loss of Ste20-like kinase induces a basal/stem-like phenotype in HER2-positive breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-05.

Duchenne's muscular dystrophy (DMD) is a severe muscle wasting disorder characterized by the loss of dystrophin expression, muscle necrosis, inflammation and fibrosis. Ongoing muscle regeneration is impaired by persistent cytokine stress, further decreasing muscle function. Patients with DMD rarely survive beyond their early 20s, with cardiac and respiratory dysfunction being the primary cause of death. Despite an increase in our understanding of disease progression as well as promising preclinical animal models for therapeutic intervention, treatment options for muscular dystrophy remain limited and novel therapeutic targets are required. Many reports suggest that the TGFβ signalling pathway is activated in dystrophic muscle and contributes to the pathology of DMD in part by impairing the differentiation of myoblasts into mature myofibers. Here, we show that in vitro knockdown of the Ste20-like kinase, SLK, can partially restore myoblast differentiation downstream of TGFβ in a Smad2/3 independent manner. In an mdx model, we demonstrate that SLK is expressed at high levels in regenerating myofibers. Muscle-specific deletion of SLK reduced leukocyte infiltration, increased myogenin and utrophin expression and enhanced differentiation. This was accompanied by resistance to eccentric contraction-induced injury in slow fiber type-enriched soleus muscles. Finally, we found that these effects were partially dependent on the upregulation of p38 signalling. Collectively, these results demonstrate that SLK downregulation can restore some aspects of disease progression in DMD.

We have previously shown that the Ste20-like kinase SLK interacts directly with the LIM domain-binding protein 1, Ldb1. Ldb1 knock down in murine fibroblasts activates SLK and enhances cell migration. To investigate the effect of Ldb1 deletion in ErbB2/HER2-driven tumorigenesis, Ldb1 conditional mice were crossed into MMTV-NIC mice, expressing the Neu oncogene and Cre recombinase from a bi-cistronic transgene. Our results show that Ldb1 is expressed in the mammary epithelium and that deletion of Ldb1 does not impair mammary gland development. Although high levels of Ldb1 can be correlated with poor prognosis in HER2+ breast cancers, Ldb1 ablation does not affect Neu-induced tumor progression in transgenic mice. Surprisingly, Ldb1 deletion did not affect SLK kinase activity in primary tumors or established cell lines. Nevertheless, Ldb1-deficient tumor cells showed enhanced mesenchymal and migratory characteristics in vitro. However, Ldb1-null cells failed to colonize the lungs of wildtype female mice when injected into the tail vein. Together our results show that Ldb1 is dispensable for mammary gland development and Neu-induced tumor progression but required for dissemination at secondary sites. Furthermore, our data also highlight contrasting cell line behaviours observed from in vivo and in vitro assays.

The Androgen Receptor (AR) has recently garnered a lot of attention as a potential biomarker and therapeutic target in hormone-dependent cancers, including breast cancer. However, several inconsistencies exist within the literature as to which subtypes of breast cancer express AR or whether it can be used to define its own unique subtype. Here, we analyze 1246 invasive breast cancer samples from the Cancer Genome Atlas and show that human breast cancers that have been subtyped based on their HER2, ESR1, or PGR expression contain four clusters of genes that are differentially expressed across all subtypes. We demonstrate that Sox10 is highly expressed in approximately one-third of all HER2/ESR1/PGR-low tumors and is a candidate biomarker of the triple-negative subtype. Although AR expression is acquired in many breast cancer cases, its expression could not define a unique subtype. Despite several reports stating that AR expression is acquired in HER2/ESR1/PGR triple-negative cancers, here we show that a low percentage of these cancers express AR (~20%). In contrast, AR is highly expressed in HER2-positive or ESR1/PGR-positive cancers (> 95%). Although AR expression cannot be used as an independent subtype biomarker, our analysis shows that routine evaluation of AR expression in tumors which express HER2, ESR1 and/or PGR may identify a unique subset of tumors which would benefit from anti-androgen based therapies.

The Ste20-like kinase, SLK, plays an important role in cell proliferation and cytoskeletal remodeling. In fibroblasts, SLK has been shown to respond to FAK/Src signaling and regulate focal adhesion turnover through Paxillin phosphorylation. Full-length SLK has also been shown to be essential for embryonic development. In myoblasts, the overexpression of a dominant negative SLK is sufficient to block myoblast fusion.

Invasion can be stimulated in vitro using the soluble ligand transforming growth factor-β (TGFβ) to induce a process called epithelial-to-mesenchymal transition (EMT) characterized by cell-cell junction breakdown and an invasive phenotype. We have previously demonstrated a role for Ste20-like kinase SLK cell migration and invasion. Here we show that SLK depletion in NMuMG mammary epithelial cells significantly impairs their TGFβ-induced migration and invasion. Immunofluorescence studies show that a fraction of SLK localizes to E-cadherin-positive adherens junction and that SLK impairs the breakdown of cell-cell contacts. We find that SLK-depleted cultures express significantly lower levels of vimentin protein as well as Snai1 and E-cadherin mRNA levels following TGF-β treatment. Surprisingly, our data show that SLK depletion does not affect the activation and nuclear translocation of Smad3. Furthermore, we show that expression of a dominant negative kinase does not impair tight junction breakdown and rescues Snai1 mRNA expression levels. Together these data suggest that SLK plays a novel role in TGFβ-induced EMT, independent of Smads, in a kinase activity-independent manner.

INTRODUCTION:Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.METHODS:Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis.RESULTS:Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn-null animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn-null environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15).CONCLUSIONS:Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover and is regulated by the LIM domain-binding proteins Ldb1 and Ldb2. These adapter proteins have been demonstrated to interact with LMO4 in the organization of transcriptional complexes. Therefore, we have assessed the ability of LMO4 to also interact and regulate SLK activity. Our data show that LMO4 can directly bind to SLK and activate its kinase activity in vitro and in vivo. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding and Cre-mediated deletion of LMO4 in conditional LMO4fl/fl fibroblasts inhibits cell migration and SLK activation. Deletion of LMO4 impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Supporting this, Src/Yes/Fyn-deficient cells (SYF) expressing very low levels of LMO4 do not recruit SLK to the leading edge. Re-expression of wildtype Myc–LMO4 in SYF cells, but not a mutant version, restores SLK localization and kinase activity. Overall, our data suggest that activation of SLK by haptotactic signals requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. Furthermore, this establishes a novel cytosolic role for the transcriptional co-activator LMO4.