Combination of Mycobacterium indicus pranii and Heat-Induced Promastigotes Cures Drug-Resistant Leishmania Infection: Critical Role of Interleukin-6-Producing Classical Dendritic Cells.

The major issues in available therapeutic modalities against leishmaniasis are cost, toxicity, and the emergence of drug resistance. The aim of this work was to develop a successful therapeutic adjuvant against drug-resistant Leishmania donovani infection by means of combining Mycobacterium indicus pranii with heat-induced promastigotes (HIP). One-month postinfected BALB/c mice were administered subcutaneously with M. indicus pranii (108 cells) and HIP (100 µg) for 5 days. Spleens were harvested for flow cytometric and reverse transcriptase PCR analysis. The antileishmanial effect of the combination strategy was associated with induction of a disease-resolving Th1 and Th17 response with simultaneous downregulation of CD4+ CD25+ Foxp3+ (nTreg) cells and CD4+ CD25- Foxp3- (Tr1) cells in the spleen. The significant expansion of CD4+ TCM (CD4+ CD44hi CD11ahi CD62Lhi) cells was a further interesting outcome of this therapeutic strategy in the context of long-term protection of hosts against secondary infection. Toll-like receptor 2 (TLR2) was also found instrumental in this antiparasitic therapy. Induced interleukin-6 (IL-6) production from expanded CD11c+ CD8α+ (cDC1) and CD11c+ CD11b+ (cDC2) dendritic cells (DCs) but not from the CD11b+ Ly6c+ inflammatory monocytes (iMOs), was found critical in the protective expansion of Th17 as evidenced by an in vivo IL-6 neutralization assay. It also promoted the hematopoietic conversion toward DC progenitors (pre-DCs) from common dendritic cell progenitors (CDPs), the immediate precursors, in bone marrow. This novel combinational strategy demonstrated that expansion of Th17 by IL-6 released from CD11c+ classical DCs is crucial, together with the conventional Th1 response, to control drug-resistant infection.

Thermal management of photonic integrated circuits: impact of holder material and epoxies.

The thermal management of photonic integrated circuits (PICs) poses a few challenges at the packaging level. The integration of a PIC with a proper holder provides mechanical support and electrical interconnection, as well as spreading of the heat generated during PIC operation. This study proposes and evaluates the thermal behavior of silicon and diamond holders and estimates the impact of integrating them with PICs as a packaged system; to this end, the thermal profile of a PIC with distributed feedback lasers mounted on silicon or diamond holders was simulated for different power levels using ANSYS Mechanical software. The impact of the epoxy resin used to mount the PIC on the holder and the thermal crosstalk between the active components were also evaluated. Based on steady-state thermal analysis, when the PIC is assembled via wire bonding, the replacement of the silicon holder with a diamond holder brings only a marginal advantage. The choice of epoxy has a larger impact on the maximum PIC temperature and the thermal crosstalk between active components. Choosing a large-thermal-conductivity-value epoxy is thus a mandatory requirement in order to guarantee the proper thermal management of PICs and their corresponding holders.

Synthesis and biological evaluation of polyhydroxylated oxindole derivatives as potential antileishmanial agent.

The devastating appearance of numerous drug-unresponsive strains of Leishmania donovani and severe toxic side effects of conventional antileishmanial therapy necessitates the search for novel leads, to treat visceral leishmaniasis efficiently. The current study deals with the synthesis and biological evaluation of a unique C-5 functionalized oxindole based polyphenol to ascertain its activities against L. donovani infection, in vitro. The polyhydroxylated oxindole derivative (1) was generated by coupling styrene derivatives with 5-bromo bis-arylidene oxindole using Heck coupling reaction. The synthesized molecule 1 was tested for its antileishmanial activity using both promastigote and amastigote stages of L. donovani. Molecule 1 showed promising anti-promastigote and anti-amastigote activities with IC50 values 15 µM and 1 µM, respectively, with no cytotoxicity towards host splenocytes. The results revealed that this compound induced parasite death by promoting oxidative stress, thereby triggering apoptosis.

Selective in vitro inhibition of Leishmania donovani by a semi-purified fraction of wild mushroom Grifola frondosa.

The current study was designed to assess the anti-leishmanial effect of a semi-purified fraction of wild mushroom Grifola frondosa against Leishmania donovani, in vitro. A total of five extracts from three wild mushrooms [Grifola frondosa (family, Meripilaceae) Laetiporus sulphurous (family, Polyporaceae) and Meripilus giganteus (family, Meripilaceae) were explored for novel anti-leishmanial leads against promastigotes. The ethanol extract of G. frondosa was selected as the most efficient against L. donovani promastigotes (IC50: 93.9 µg/mL). A semi-purified fraction was obtained from an active ethanol extract of G. frondosa and found to inhibit the survival of promastigotes of L. donovani (MHOM/IN/83/AG83) significantly (IC50: 20.37 µg/mL) and it also had some effect against L. major LV39 (MRHO/Sv/59/P strain) and L. tropica WR683 (MHOM/SU/58/OD) strains at higher concentrations (IC50: 46.08 µg/mL and 53.79 µg/mL respectively). The semi-purified fraction also interfered in lipid biosynthesis, altered parasite morphology and induced apoptosis in L. donovani promastigotes. The semi-purified fraction was also effective against intracellular amastigotes in infected macrophages and enhanced the release of nitric oxide and pro-inflammatory cytokines, in vitro. Interestingly, the 50% inhibitory concentration of the semi-purified fraction against the intracellular amastigotes (IC50: 2.48 µg/mL) was much lower in comparison to promastigotes (IC50: 20.37 µg/mL). The semi-purified fraction was found to inhibit the intracellular amastigotes slightly more efficiently in comparison to conventional anti-leishmanial drugs; sodium antimony gluconate, amphotericin B, miltefosine and paromomycin and noticeably non-toxic towards host splenocytes. The findings of the present study established that G. frondosa might be a natural resource for development of a new anti-leishmanial lead.

Successful Therapy of Murine Visceral Leishmaniasis with Astrakurkurone, a Triterpene Isolated from the Mushroom Astraeus hygrometricus, Involves the Induction of Protective Cell-Mediated Immunity and TLR9.

In our previous report, we showed that astrakurkurone, a triterpene isolated from the Indian mushroom Astraeus hygrometricus (Pers.) Morgan, induced reactive oxygen species, leading to apoptosis in Leishmania donovani promastigotes, and also was effective in inhibiting intracellular amastigotes at the 50% inhibitory concentration of 2.5 µg/ml. The aim of the present study is to characterize the associated immunomodulatory potentials and cellular activation provided by astrakurkurone, leading to effective antileishmanial activity in vitro and in vivo Astrakurkurone-mediated antileishmanial activity was evaluated by real-time PCR and flow cytometry. The involvement of Toll-like receptor 9 (TLR9) was studied by in vitro assay in the presence of a TLR9 agonist and antagonist and by in silico modeling of a three-dimensional structure of the ectodomain of TLR9 and its interaction with astrakurkurone. Astrakurkurone caused a significant increase in TLR9 expression of L. donovani-infected macrophages along with the activation of proinflammatory responses. The involvement of TLR9 in astrakurkurone-mediated amastigote killing has been evidenced from the fact that a TLR9 agonist (CpG, ODN 1826) in combination with astrakurkurone enhanced the amastigote killing, while a TLR9 antagonist (bafilomycin A1) alone or in combination with astrakurkurone curbed the amastigote killing, which could be further justified by in silico evidence of docking between mouse TLR9 and astrakurkurone. Astrakurkurone was found to reduce the parasite burden in vivo by inducing protective cytokines, gamma interferon and interleukin 17. Moreover, astrakurkurone was nontoxic toward peripheral blood mononuclear cells of immunocompromised patients with visceral leishmaniasis. Astrakurkurone, a nontoxic antileishmanial, enhances the immune efficiency of host cells, leading to parasite clearance in vitro and in vivo.

Bem3, a Cdc42 GTPase-activating protein, traffics to an intracellular compartment and recruits the secretory Rab GTPase Sec4 to endomembranes.

Cell polarity is essential for many cellular functions including division and cell-fate determination. Although RhoGTPase signaling and vesicle trafficking are both required for the establishment of cell polarity, the mechanisms by which they are coordinated are unclear. Here, we demonstrate that the yeast RhoGAP (GTPase activating protein), Bem3, is targeted to sites of polarized growth by the endocytic and recycling pathways. Specifically, deletion of SLA2 or RCY1 led to mislocalization of Bem3 to depolarized puncta and accumulation in intracellular compartments, respectively. Bem3 partitioned between the plasma membrane and an intracellular membrane-bound compartment. These Bem3-positive structures were polarized towards sites of bud emergence and were mostly observed during the pre-mitotic phase of apical growth. Cell biological and biochemical approaches demonstrated that this intracellular Bem3 compartment contained markers for both the endocytic and secretory pathways, which were reminiscent of the Spitzenkörper present in the hyphal tips of growing fungi. Importantly, Bem3 was not a passive cargo, but recruited the secretory Rab protein, Sec4, to the Bem3-containing compartments. Moreover, Bem3 deletion resulted in less efficient localization of Sec4 to bud tips during early stages of bud emergence. Surprisingly, these effects of Bem3 on Sec4 were independent of its GAP activity, but depended on its ability to efficiently bind endomembranes. This work unveils unsuspected and important details of the relationship between vesicle traffic and elements of the cell polarity machinery: (1) Bem3, a cell polarity and peripherally associated membrane protein, relies on vesicle trafficking to maintain its proper localization; and (2) in turn, Bem3 influences secretory vesicle trafficking.

Protective effect of Croton caudatus Geisel leaf extract against experimental visceral leishmaniasis induces proinflammatory cytokines in vitro and in vivo.

In the present state of overwhelming emergence of drug-unresponsive phenotypes of Leishmania donovani and persistent severe toxicity in conventional anti-leishmanial therapy, in search for novel leads, the aim of this study has been fixed to identify the active extract(s) of Croton caudatus Geisel. var. tomentosus Hook effective against the parasitic protozoans in vitro and in vivo. C. caudatus Geisel. is often used by Chakma and Hmar community, the local tribes of north-east India for medicinal and veterinary purposes. Among the five semi-purified extracts tested, C. caudatus leaves, extracted in hexane and subsequently semi-purified in a column packed with silica gel (70-130 µM; mesh size 60 A°) using ethyl acetate-hexane solvent (9:1), was found to be the most effective growth inhibitor (JDHex) against the Leishmania promastigotes and amastigotes. JDHex significantly altered the biochemical parameters (protein, lipid and carbohydrates) in promastigotes followed by the morphological changes, DNA condensation and subsequent apoptosis in L. donovani. In consequent steps, it has been also proved that JDHex reduced the replication of intracellular amastigotes with concomitant release of nitric oxide and pro-inflammatory cytokines, IL-12 and TNF-α in vitro. Significantly, the 50% inhibitory concentration of JDHex was estimated much lower against the intracellular amastigotes (2.5 µg/mL) in comparison to promastigotes (10 µg/mL). JDHex was also found efficient in reducing parasite burden in spleen and liver when treated in vivo and increased the intracellular IFN-γ and decreased the IL-10 in CD4+ T cells in splenocytes of orally treated animals. The results of this study support the importance in exploration of novel anti-leishmanial leads from C. caudatus Geisel. var. tomentosus Hook. against the L. donovani (MHOM/IN/83/AG83) infection. Partial chemical characterization of JDHex revealed the presence of terpenoids. However, the further chemical investigation of JDHex is warranted.

The Lowe syndrome protein OCRL1 is involved in primary cilia assembly.

Lowe syndrome (LS) is a devastating, X-linked genetic disease characterized by the presence of congenital cataracts, profound learning disabilities and renal dysfunction. Unfortunately, children affected with LS often die early of health complications including renal failure. Although this syndrome was first described in the early 1950s and the affected gene, OCRL1, was identified more than 17 years ago, the mechanism by which Ocrl1 defects lead to LS's symptoms remains unknown. Here we show that LS display characteristics of a ciliopathy. Specifically, we found that patients' cells have defects in the assembly of primary cilia and this phenotype was reproduced in cell lines by knock-down of Ocrl1. Importantly, this defect could be rescued by re-introduction of WT Ocrl1 in both patient and Ocrl1 knock-down cells. In addition, a zebrafish animal model of LS exhibited cilia defects and multiple morphological and anatomical abnormalities typically seen in ciliopathies. Mechanistically, we show that Ocrl1 is involved in protein trafficking to the primary cilia in an Rab8-and IPIP27/Ses-dependent manner. Taking into consideration the relevance of the signaling pathways hosted by the primary cilium, our results suggest hitherto unrecognized mechanisms by which Ocrl1 deficiency may contribute to the phenotypic characteristics of LS. This conceptual change in our understanding of the disease etiology may provide an alternative avenue for the development of therapies.

Selective inhibition of Leishmania donovani by active extracts of wild mushrooms used by the tribal population of India: An in vitro exploration for new leads against parasitic protozoans.

The study was intended at evaluating the anti-proliferating effect of mushrooms used in traditional folklore of Santal tribal population in India against Leishmania donovani (MHOM/IN/83/AG83). A total of eighteen extracts, three estracts from each mushroom [(80% ethanol extracted; Fa), (water-soluble polysaccharide fraction; Fb), (polyphenolic fraction; Fc)], from six wild mushrooms were obtained. These extracts were tested against the promastigotes and amastigotes for their antileishmanial capacity. Fa fractions (250 µg/mL) of Astraeus hygrometricus and Tricholoma giganteum significantly inhibited the growth of L. donovani promastigotes and interfered in lipid biosynthesis. Moreover, both fractions induced apoptosis in promastigotes. Water soluble Fb fractions of A. hygrometricus, Russula laurocerasi, Russula albonigra, Termitomyces eurhizus, Russula delica and polyphenolic Fc fraction of R. laurocerasi were found to inhibit the replication of intracellular amastigotes in macrophages dose dependently. Significantly, 50% inhibitory concentration of the active extracts against intracellular amastigotes induced release of nitric oxide and IL-12 in murine macrophages and dendritic cells assay and also found considerably non-toxic on murine splenocytes. Results of this study can be used as a basis for further phytochemical and pharmacological investigations in the effort for search of novel anti-leishmanial leads.

Prevention of arthritis markers in experimental animal and inflammation signalling in macrophage by Karanjin isolated from Pongamia pinnata seed extract.

Karanjin, the furanoflavonoid reported to possess gastroprotective and anti-diabetic properties, was investigated against experimental arthritis and its molecular signalling in inflammation was explored in macrophages. Karanjin was isolated from hexane extract of Pongamia pinnata seeds and was evaluated on arthritis markers in adjuvant induced arthritis model (AIA) in two doses (per oral; 10 mg/kg/day and 20 mg/kg/day). Karanjin dose dependently reduced collagen and cartilage breakdown markers viz. urinary hydroxyproline and glucosamine, respectively, serum lysosomal enzymes responsible for articular cartilage damage, and major proinflammatory cytokine TNFα, secreted by macrophages involved in articular inflammation and destruction. Karanjin also prevented joint damage as evidenced from arthritis score, radiographic and histopathological analysis. To delineate the molecular target of Karanjin, in vitro study on LPS induced macrophages were performed at calibrated non toxic doses (4 µg/mL and 6 µg/mL). Karanjin reduced TNFα production and also showed potent inhibitory effect on nitric oxide and reactive oxygen species production which is generally induced by TNFα from activated macrophages. NF-κB, the key regulator of TNFα signalling during inflammation was significantly suppressed by Karanjin. Our study for the first time highlights the anti-inflammatory role of Karanjin in experimental arthritis model as well as on macrophage signalling, thereby depicting its probable mechanism of action.

Practices and outcomes of responsive caregiving on child neurodevelopment and mental health across diverse global populations: a scoping review protocol.

INTRODUCTION: Responsive caregiving (RC) leads to positive outcomes in children, including secure attachment with caregivers, emotional regulation, positive social interactions and cognitive development. Through our scoping review, we aim to summarise the practices and outcomes of RC in diverse caregiver and child populations from 0 to 8 years. METHODS AND ANALYSIS: We will use the Arksey and O'Malley framework and the Joanna Briggs Institute methodology for scoping reviews. We shall present our findings as per the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines for scoping review. Only peer-reviewed, English-language articles from 1982 to 2022 will be included from PubMed, Web of Science, APA PsychInfo, APA PsycArticles, SocINDEX and Google Scholar databases. Reference lists of included articles will also be screened. The search strategy will be developed for each database, and search results will be imported into Rayyan. Screening will be done in two phases: (1) titles and abstracts will be screened by two authors and conflicts will be resolved by mutual discussion between both or by consulting with a senior author; and (2) full-texts of shortlisted studies from the first phase will then be screened using the same inclusion/exclusion criteria. A data extraction form will be developed to collate relevant information from the final list of included articles. This form will be pilot tested on the first 10 papers and iteratively refined prior to data extraction from the remaining articles. Results will be presented in figures, tables and a narrative summary. ETHICS AND DISSEMINATION: No ethics approval needed as the review shall only use already published data. We shall publish the review in an open-access, peer-reviewed journal and disseminate through newsletters, social media pages, and presentations to relevant audiences.

Using mobile health technology to assess childhood autism in low-resource community settings in India: An innovation to address the detection gap.

LAY ABSTRACT: Autism is diagnosed by highly trained professionals- but most autistic people live in parts of the world that harbour few or no such autism specialists and little autism awareness. So many autistic people go undiagnosed, misdiagnosed, and misunderstood. We designed an app (START) to identify autism and related conditions in such places, in an attempt to address this global gap in access to specialists. START uses computerised games and activities for children and a questionnaire for parents to measure social, sensory, and motor skills. To check whether START can flag undiagnosed children likely to have neurodevelopmental conditions, we tested START with children whose diagnoses already were known: Non-specialist health workers with just a high-school education took START to family homes in poor neighbourhoods of Delhi, India to work with 131 two-to-seven-year-olds. Differences between typically and atypically developing children were highlighted in all three types of skills that START assesses: children with neurodevelopmental conditions preferred looking at geometric patterns rather than social scenes, were fascinated by predictable, repetitive sensory stimuli, and had more trouble with precise hand movements. Parents' responses to surveys further distinguished autistic from non-autistic children. An artificial-intelligence technique combining all these measures demonstrated that START can fairly accurately flag atypically developing children. Health workers and families endorsed START as attractive to most children, understandable to health workers, and adaptable within sometimes chaotic home and family environments. This study provides a proof of principle for START in digital screening of autism and related conditions in community settings.

Scalable Transdiagnostic Early Assessment of Mental Health (STREAM): a study protocol.

INTRODUCTION: Early childhood development forms the foundations for functioning later in life. Thus, accurate monitoring of developmental trajectories is critical. However, such monitoring often relies on time-intensive assessments which necessitate administration by skilled professionals. This difficulty is exacerbated in low-resource settings where such professionals are predominantly concentrated in urban and often private clinics, making them inaccessible to many. This geographic and economic inaccessibility contributes to a significant 'detection gap' where many children who might benefit from support remain undetected. The Scalable Transdiagnostic Early Assessment of Mental Health (STREAM) project aims to bridge this gap by developing an open-source, scalable, tablet-based platform administered by non-specialist workers to assess motor, social and cognitive developmental status. The goal is to deploy STREAM through public health initiatives, maximising opportunities for effective early interventions. METHODS AND ANALYSIS: The STREAM project will enrol and assess 4000 children aged 0-6 years from Malawi (n=2000) and India (n=2000). It integrates three established developmental assessment tools measuring motor, social and cognitive functioning using gamified tasks, observation checklists, parent-report and audio-video recordings. Domain scores for motor, social and cognitive functioning will be developed and assessed for their validity and reliability. These domain scores will then be used to construct age-adjusted developmental reference curves. ETHICS AND DISSEMINATION: Ethical approval has been obtained from local review boards at each site (India: Sangath Institutional Review Board; All India Institute of Medical Science (AIIMS) Ethics Committee; Indian Council of Medical Research-Health Ministry Screening Committee; Malawi: College of Medicine Research and Ethics Committee; Malawi Ministry of Health-Blantyre District Health Office). The study adheres to Good Clinical Practice standards and the ethical guidelines of the 6th (2008) Declaration of Helsinki. Findings from STREAM will be disseminated to participating families, healthcare professionals, policymakers, educators and researchers, at local, national and international levels through meetings, academic journals and conferences.

A novel role of Krüppel-like factor 8 in DNA repair in breast cancer cells.

Krüppel-like factor 8 (KLF8) regulates critical gene transcription and cellular events associated with cancer. However, the role of KLF8 in cancer remains largely unknown. Here, we report a surprisingly novel role for KLF8 in DNA repair in breast cancer cells. Comet, clonogenic, and WST-1 assays showed that KLF8 expression is required for protecting human breast cancer cells from doxorubicin-induced DNA damage and cell death. Western blotting indicated that overexpression of ectopic KLF8 attenuated the levels of the DNA damage marker γH2A.X in doxorubicin-treated PARP-1(+/+) but not PARP-1(-/-) mouse embryonic fibroblasts, whereas the PARP-1-binding-defective KLF8 mutant failed to do so. Interestingly, in response to the DNA damage, KLF8 was phosphorylated by the DNA-dependent protein kinase catalytic subunit and, subsequently, SUMOylated by SUMO E3 ligases protein inhibitors of activated STAT (PIASs), which depends upon the interaction of KLF8 with DNA-dependent protein kinase catalytic subunit, PIASs, and PARP-1 as well as their enzymatic activities. Lastly, we show evidence that KLF8 was recruited to the DNA damage site. These results suggest a novel role and mechanism for KLF8 in the regulation of DNA repair and therapeutic resistance in breast cancer cells.

The current landscape and future of tablet-based cognitive assessments for children in low-resourced settings.

Interest in measuring cognition in children in low-resourced settings has increased in recent years, but options for cognitive assessments are limited. Researchers are faced with challenges when using existing assessments in these settings, such as trained workforce shortages, less relevant testing stimuli, limitations of proprietary assessments, and inadequate parental knowledge of cognitive milestones. Tablet-based direct child assessments are emerging as a practical solution to these challenges, but evidence of their validity and utility in cross-cultural settings is limited. In this overview, we introduce key concepts of this field while exploring the current landscape of tablet-based assessments for low-resourced settings. We also make recommendations for future directions of this relatively novel field. We conclude that tablet-based assessments are an emerging and promising method of assessing cognition in young children. Further awareness and dissemination of validated tablet-based assessments may increase capacity for child development research and clinical practice in low-resourced settings.

Increased CaMKK2 Expression Is an Adaptive Response That Maintains the Fitness of Tumor-Infiltrating Natural Killer Cells.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been interest in developing CaMKK2 inhibitors as potential anticancer therapeutics. One impediment to clinical development of these agents is that the roles of CaMKK2 in other cellular compartments within the tumor immune microenvironment remain to be established. We report herein that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. NK cell-intrinsic deletion of CaMKK2 increased metastatic progression in several murine models, establishing a critical role for this enzyme in NK cell-mediated antitumor immunity. Ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK-cell survival. These results indicate an important scaffolding function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDD) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactic acid-rich tumor microenvironment. The findings of this study should inform strategies to manipulate the CaMKK2-signaling axis as a therapeutic approach in cancer.

Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis.

Triple-negative breast cancers (TNBC) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early-stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A, which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Furthermore, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. SIGNIFICANCE: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.

Ca 2+ /Calmodulin Dependent Protein Kinase Kinase-2 (CaMKK2) promotes Protein Kinase G (PKG)-dependent actin cytoskeletal assembly to increase tumor metastasis.

Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

SGC-CAMKK2-1: A Chemical Probe for CAMKK2.

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

Identification of Small Molecule Inhibitors and Ligand Directed Degraders of Calcium/Calmodulin Dependent Protein Kinase Kinase 1 and 2 (CaMKK1/2).

CaMKK2 signals through AMPK-dependent and AMPK-independent pathways to trigger cellular outputs including proliferation, differentiation, and migration, resulting in changes to metabolism, bone mass accrual, neuronal function, hematopoiesis, and immunity. CAMKK2 is upregulated in tumors including hepatocellular carcinoma, prostate, breast, and gastric cancer, and genetic deletion in myeloid cells results in increased antitumor immunity in several syngeneic models. Validation of the biological roles of CaMKK2 has relied on genetic deletion or small molecule inhibitors with activity against several biological targets. We sought to generate selective inhibitors and degraders to understand the biological impact of inhibiting catalytic activity and scaffolding and the potential therapeutic benefits of targeting CaMKK2. We report herein selective, ligand-efficient inhibitors and ligand-directed degraders of CaMKK2 that were used to probe immune and tumor intrinsic biology. These molecules provide two distinct strategies for ablating CaMKK2 signaling in vitro and in vivo.