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In the file an additional information on the screen workflow and characterization of the effects of the genes that were found in the screen on invadopodia formation. Further there is Supplementary Table 1 (screen results for 63T cells). In addition, there are supplementary experiments supporting the role of AXL as a regulator of invadopodia in melanoma, as well as breast cancer cells.

Statistics for gene expression analysis (Figure 6A). In the first tab, significant levels of ERRB3 and AXL expression against all 6 genes (AXL, ERBB3, MITF3 and the 3 targets) in each cluster. In the second tab, significant levels of differences in invadopodia genes between Clusters 1 and 2, as well as the delta median of expression for each gene. In the third tab, significant levels of the differences in TK genes between Clusters 1 and 2, as well as the delta median of expression for each gene.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma.Significance:These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.

In the Excel file, the raw data for the screens (3 in A375 cells, and 2 in 63T cells). Every tab contains one screen and all the genes that were tested. The data for each screen are quantified, and the results, summarized.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.

Cancer immunotherapy with immune checkpoint blockade (ICB) has transformed the treatment of melanoma, although intrinsic or acquired resistance develops in nearly half of patients. Tumor-infiltrating CD8+ T lymphocytes (TILs) are key determinants of anti-tumor immunity in melanoma and other cancers, and single-cell RNA-sequencing has identified T cell states associated with improved clinical response to ICB, as well as adoptive T cell therapy (ACT). Despite these advances, strategies to identify and analyze tumor-reactive TILs in ICB-resistant patients remain limited. Here, we demonstrate that TILs from ICB-resistant melanoma patients can recognize and eliminate autologous tumor cells independent of class I HLA-TCR interactions. TILs eliminated matched melanoma cells in a time and dose-dependent fashion associated with secretion of effector cytokines. Strikingly, the deletion of B2M (resulting in loss of class I HLA surface expression) did not alter the activity of these TILs. Immunophenotyping studies confirmed that TILs are largely (>95%) effector memory (Tem) CD8 T cells (CD45RA-CD45RO+CCR7-) and give rise to terminal effector cells after co-culture with matched melanoma cells. Further, the elimination of melanoma cells by TILs required intact JAK1/2 signaling, although interferon-gamma (IFNγ) was neither necessary nor sufficient for tumor cell elimination. Together, these findings demonstrate that expanded TILs from ICB-resistant melanoma patients are capable of eliminating melanoma cells via a novel, class I MHC-independent mechanism. Citation Format: Hongyan Xie, Aiping Jiang, Anne Jenney, Yi Sun, Tatyana Sharova, Moshe Sade-Feldman, Or-yam Revach, Angelina Cicerchia, Martin Q. Rasmussen, Nir Hacohen, Robert T. Manguso, Russell W. Jenkins. Class I HLA-independent lysis of immunotherapy-resistant melanoma by CD8 T cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4072.

Despite the unprecedented success of immune checkpoint blockade (ICB) in melanoma and other cancers, therapeutic resistance remains a major challenge. CD38, an ecto-enzyme involved in NAD+ catabolism, is upregulated in dysfunctional/exhausted CD8+ T cells in human melanoma. As enrichment of dysfunctional CD8+ T cells is associated with lack of response to ICB, and NAD+ depletion via enhanced CD38 activity has been associated with diminished immune response, we hypothesized that CD38 might represent an attractive target to overcome resistance to ICB. While CD38 blockade has been shown to augment response to ICB in murine tumor models, the specific role of CD38 signaling in CD8 T cell exhaustion, and the therapeutic relevance of CD38 in human cancer have not been defined. Here, we confirm and extend previous observations that CD38 is enriched in exhausted CD8+ T cells during tumor progression and in response to ICB. Further, we demonstrate that disrupting CD38 signaling via neutralizing antibodies or pharmacological inhibition enhances response to ICB using established murine tumor models and patient-derived tumor explant models. Notably, we observed 54% response rate to combination PD-1/CD38 blockade using a cohort (n=35) of patient-derived organotypic tumor spheroids (PDOTS) from patients with ICB-refractory melanoma (11.4% response rate to single-agent PD-1 blockade). Supplementation of nicotinamide riboside (NR) to boost NAD+ levels mimicked the effects CD38 blockade/inhibition on PD-1 response, whereas FK866, an inhibitor of NAMPT a key step in NAD+ biosynthesis, blunted the response to combined PD-1/CD38 blockade, suggesting a major role for NAD+ in the efficacy of CD38 blockade/inhibition. Lastly, we demonstrate that in vitro CD38 inhibition/blockade in CD8+ T cells increases the levels of TCF7 (an effector/memory T cell transcription factor), decreases surface expression of co-inhibitory receptors (e.g., TIM-3, CD39, PD-1), and improves effector CD8 T cell function. Taken together, these data confirm a role for CD38 in CD8+ T cell exhaustion in melanoma and support further pre-clinical and clinical development of this novel therapeutic strategy to enhance anti-tumor immune responses. Citation Format: Or-Yam Revach, Angelina M. Cicerchia, Moshe Sade-Feldman, Seth Anderson, Robert T. Manguso, Nir Hacohen, Russell W. Jenkins. Targeting CD38 on exhausted T cells overcomes resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3250.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking. We identified the innate immune kinase TANK-binding kinase 1 (TBK1) as a candidate immune evasion gene in a pooled genetic screen. Using a suite of genetic and pharmacologic tools across multiple experimental model systems, we confirm a role for TBK1 as an immune evasion gene. Targeting TBK1 enhances response to PD-1 blockade by lowering the cytotoxicity threshold to effector cytokines (TNFα/IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumor models. Cancer cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNFα/IFNγ in a JAK/STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is a novel and effective strategy to overcome resistance to cancer immunotherapy. Citation Format: Yi Sun, Or-Yam Revach, Seth Anderson, Anne Jenney, Caitlin E. Mills, Payal Tiwari, Robert T. Manguso, Russell William Jenkins. Targeting TBK1 to overcome resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3285.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified a series of regulators, the knockdown of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances (e.g., ABL2, AXL, CSK) invadopodia formation and function. Notably, the receptor tyrosine kinase AXL displayed a dual regulatory function, where both depletion or overexpression enhanced invadopodia formation and activity. This apparent contradiction was attributed to the capacity of AXL to directly stimulate invadopodia, yet its suppression upregulates the ERBB3 signaling pathway, which can also activate core invadopodia regulators and enhance invadopodia function. Bioinformatic analysis of multiple melanoma cell lines points to an inverse expression pattern of AXL and ERBB3. High expression of AXL in melanoma cells is associated with high expression of invadopodia components and an invasive phenotype. These results provide new insights into the complexity of metastasis-promoting mechanisms and suggest that targeting of multiple invadopodia signaling networks may serve as a potential anti-invasion therapy in melanoma. Significance: These findings uncover a unique interplay between AXL and ERBB3 in invadopodia regulation that points to the need for combined therapy in order to prevent invadopodia-mediated metastasis in melanoma.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf ‘decision-sensing-system’ controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3’UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape., The chemokine CXCL10 is associated with pathogenesis of cerebral malaria in Plasmodium falciparum infection. Here the authors show that P. falciparum produces extracellular vesicles laden with RNAs that are taken up by monocytes resulting in a RIG-I and HUR-1 mediated mechanism of inhibition of CXCL10 protein translation.

Integrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling ‘adhesome’ components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different – often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their ‘adhesive’ and ‘invasive’ functionalities require distinct sources of biomechanical reinforcement.

INTRODUCTION: TANK-binding kinase 1 (TBK1) is a Ser/Thr kinase with a central role in coordinating the cellular response to invading pathogens and regulating key inflammatory signaling cascades. While intact TBK1 signaling is required for successful anti-viral signaling, dysregulated TBK1 signaling has been linked to a variety of pathophysiologic conditions, including cancer. Several lines of evidence support a role for TBK1 in cancer pathogenesis, but the specific roles and regulation of TBK1 remain incompletely understood. A key challenge is the diversity of cellular processes that are regulated by TBK1, including inflammation, cell cycle, autophagy, energy homeostasis, and cell death. Nevertheless, evidence from pre-clinical cancer models suggest that targeting TBK1 may be an effective strategy for anti-cancer therapy in specific settings. AREAS COVERED: This review provides an overview of the roles and regulation of TBK1 with a focus on cancer pathogenesis and drug targeting of TBK1 as an anti-cancer strategy. Relevant literature was derived from a PubMed search encompassing studies from 1999 to 2020. EXPERT OPINION: TBK1 is emerging as a potential target for anti-cancer therapy. Inhibition of TBK1 alone may be insufficient to restrain the growth of most cancers, hence combination strategies will likely be necessary. Improved understanding of tumor-intrinsic and tumor-extrinsic TBK1 signaling will inform novel therapeutic strategies.

SummaryCardiomyocyte (CM) loss after injury results in adverse remodelling and fibrosis, which inevitably lead to heart failure. ERBB2-Neuregulin and Hippo-YAP signaling pathways are key mediators of CM proliferation and regeneration, yet the crosstalk between these pathways is unclear. Here, we demonstrate in adult mice that transient over-expression (OE) of activated ERBB2 in CMs promotes cardiac regeneration in a heart failure model. OE CMs present an EMT-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration, and ECM turnover. Molecularly, we identified YAP as a critical mediator of ERBB2 signaling. In OE CMs, YAP interacts with nuclear envelope and cytoskeletal components, reflecting the altered mechanic state elicited by ERBB2. Hippo-independent activating phosphorylation on YAP at S352 and S274 were enriched in OE CMs, peaking during metaphase, and viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we demonstrate a potent ERBB2-mediated YAP mechanosensory signaling, involving EMT-like characteristics, resulting in heart regeneration.HighlightsERBB2-driven regeneration of scarred hearts recapitulates core-EMT processesYAP is activated and required downstream to ERBB2 signaling in CMsYAP activity is mechanically driven by cytoskeleton and nuclear envelope remodelingYAP S274 and S352 phosphorylation is essential for CM mitosis

In this article, we discuss the complex involvement of a Rho-family GTPase, Rac1, in cell migration and in invadopodia-mediated matrix degradation. We discuss the involvement of invadopodia in invasive cell migration, and their capacity to promote cancer metastasis. Considering the regulation of invadopodia formation, we describe studies that demonstrate the role of Rac1 in the metastatic process, and the suggestion that this effect is attributable to the capacity of Rac1 to promote invadopodia formation. This notion is demonstrated here by showing that knockdown of Rac1 in melanoma cells expressing a wild-type form of this GTPase, reduces invadopodia-dependent matrix degradation. Interestingly, we also show that excessive activity of Rac1, displayed by the P29S, hyperactive, “fast cycling” mutant of Rac1, which is present in 5–10% of melanoma tumors, inhibits invadopodia function. Moreover, knockdown of this hyperactive mutant enhanced matrix degradation, indicating that excessive Rac1 activity by this mutant can negatively regulate invadopodia formation and function.

The invasive phenotype of metastatic cancer cells is accompanied by the formation of actin-rich invadopodia, which adhere to the extracellular matrix, and degrade it. In this study, we explored the role of the tyrosine kinome in the formation of invadopodia in metastatic melanoma cells. Using a microscopy-based siRNA screen, we identified novel invadopodia regulators, the knock-down of which either suppresses (e.g., TYK2, IGFR1, ERBB3, TYRO3, FES, ALK, PTK7) or enhances invadopodia formation and function (e.g., ABL2, AXL, CSK). Particularly intriguing was the discovery that the receptor tyrosine kinase AXL displays a dual regulatory function, manifested by enhancement of invadopodia function upon knock-down or long-term inhibition, as well as following its over-expression. We show here that this apparent contradiction may be attributed to the capacity of AXL to directly stimulate invadopodia; yet its suppression up-regulates the ERBB3 signaling pathway, which consequently activates core invadopodia regulators, and greatly enhances invadopodia function. Bioinformatic analysis of multiple melanoma cells points to an inverse expression pattern of AXL and ERBB3, with the apparent association of high-AXL melanomas, with high expression of invadopodia components and an invasive phenotype. The relevance of these results to melanoma metastasis in vivo, and to potential anti-invasion therapy, is discussed.

Invadopodia are actin-rich membrane protrusions through which cells adhere to the extracellular matrix and degrade it. In this study, we explored the mechanical interactions of invadopodia in melanoma cells, using a combination of correlative light and electron microscopy. We show here that the core actin bundle of most invadopodia interacts with integrin-containing matrix adhesions at its basal end, extends through a microtubule-rich cytoplasm and at its apical end, interacts with the nuclear envelope and indents it. Abolishment of invadopodia by microtubules or src inhibitors leads to the disappearance of these nuclear indentations. Based on the indentation profile and the viscoelastic properties of the nucleus, the force applied by invadopodia is estimated to be in the nanoNewton range. We further show that knockdown of the LINC complex components nesprin 2 or SUN1 leads to a substantial increase in the prominence of the adhesion domains at the opposite end of the invadopodia. We discuss this unexpected, long-range mechanical interplay between the apical and basal domains of invadopodia and its possible involvement in the penetration of invadopodia into the matrix.