Druggable Kinase Research Articles

MAP3K8 is a potential therapeutic target in airway epithelial inflammation

BackgroundWe have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1β stimulation. We analysed signalling transduction and global gene expression after IL-1β stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models.ResultsIL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1β following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose.ConclusionsThese results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.

Investigating druggable kinases for targeted therapy in retinoblastoma.

Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.

Abstract B007: Vandetanib abrogates antiestrogen resistance in MAPK-driven ER+ breast cancer by halting key intracellular signaling pathways and multiple druggable kinases

Abstract Introduction: Vandetanib has been shown to abrogate ER+ breast cancer growth via inhibition of MAPK; we sought to elucidate the effects of Vandetanib on MAPK-driven endocrine-resistant breast cancer. Methods: Chronically treated MCF7-TAM (Tamoxifen), MCF7-EXE (Exemestane), and wild-type MCF7 ER+ breast cancer cell lines were treated with Vandetanib and/or Tamoxifen. Cell viability assays were conducted with CellTiter-Glo. Cells were treated with Vandetanib (10 uM) and vehicle for 24 hours, and mRNA sequencing was performed. To elicit TKI-specific proteomic changes, cells were treated with control or IC90 Vandetanib for 48 hours, and the signatures of kinase activity were determined using a multiplex inhibitor bead library coupled with mass spectrometry. Differential gene (DGE) and protein (DPE) expression analyses were performed to determine enriched and depleted transcripts and kinase activity with Vandetanib treatment. Gene set enrichment analysis was performed to determine the shared and unique pathways regulated by Vandetanib for each model. A Vandetanib response signature was compiled using the top 20 downregulated genes and analyzed with METABRIC and SCAN-B datasets to assess effects on survival in ER+ breast cancer patients. Results: MCF7-TAM and MCF7-EXE demonstrated lower Tamoxifen IC50 when treated with Vandetanib compared to Tamoxifen alone, which showed synergistic effects. Data analysis of mRNA libraries showed reduced tumor progression and signaling of MAPK, PI3K/AKT, RAS, CCND1, MYC, and EGFR across all three lines, as well as increased ERBB2 signaling, which may denote a potential adaptive bypass mechanism to Vandetanib treatment. Gene sets showed increased estrogen response, ESR1 activity, and sensitivity to tamoxifen across all three lines. Notable key players in PI3K/AKT and MAPK signaling such as TYRO3 were significantly downregulated at both the gene and protein levels. Additionally, JAK2 demonstrated dual upregulation and has been implicated with favorable prognosis due to associations with immune infiltration. The downregulated Vandetanib gene set conferred significantly improved OS (HR=0.75, p&amp;lt;0.0001, METABRIC; HR=0.87, p=0.034, SCAN-B) and RFS (HR=0.60, p&amp;lt;0.0001, METABRIC) in ER+ breast cancer patients from both datasets. Multiple druggable kinases such as RET, EGFR, FGFR2/3, CDK6, and SRC were depleted by Vandetanib treatment. Upregulated druggable kinases identified with Vandetanib treatment include MTOR, FGFR4, ERBB2, and JAK1. Conclusions: Vandetanib overcomes antiestrogen resistance in MAPK-driven models of ER+ breast cancer by halting key signaling pathways such as MAPK, PI3K/AKT, MYC, and ERBB2, among others. Upregulated druggable kinases may serve as targets to overcome resistance to Vandetanib. Downregulated targets with Vandetanib could serve as biomarkers to select patients most likely to benefit from Vandetanib treatment. Citation Format: Rasha T Kakati, Austin Whitman, Hyunsoo Kim, Philip M Spanheimer. Vandetanib abrogates antiestrogen resistance in MAPK-driven ER+ breast cancer by halting key intracellular signaling pathways and multiple druggable kinases [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B007.

Comprehensive split TEV based protein-protein interaction screening reveals TAOK2 as a key modulator of Hippo signalling to limit growth.

The conserved Hippo signalling pathway plays a crucial role in tumour formation by limiting tissue growth and proliferation. At the core of this pathway are tumour suppressor kinases STK3/4 and LATS1/2, which limit the activity of the oncogene YAP1, the primary downstream effector. Here, we employed a split TEV-based protein-protein interaction screen to assess the physical interactions among 28 key Hippo pathway components and potential upstream modulators. This screen led us to the discovery of TAOK2 as pivotal modulator of Hippo signalling, as it binds to the pathway's core kinases, STK3/4 and LATS1/2, and leads to their phosphorylation. Specifically, our findings revealed that TAOK2 binds to and phosphorylates LATS1, resulting in the reduction of YAP1 phosphorylation and subsequent transcription of oncogenes. Consequently, this decrease led to a decrease in cell proliferation and migration. Interestingly, a correlation was observed between reduced TAOK2 expression and decreased patient survival time in certain types of human cancers, including lung and kidney cancer as well as glioma. Moreover, in cellular models corresponding to these cancer types the downregulation of TAOK2 by CRISPR inhibition led to reduced phosphorylation of LATS1 and increased proliferation rates, supporting TAOK2's role as tumour suppressor gene. By contrast, overexpression of TAOK2 in these cellular models lead to increased phospho-LATS1 but reduced cell proliferation. As TAOK2 is a druggable kinase, targeting TAOK2 could serve as an attractive pharmacological approach to modulate cell growth and potentially offer strategies for combating cancer.

Mutability of druggable kinases and pro-inflammatory cytokines by their proximity to telomeres and A+T content.

Mutations of protein kinases and cytokines are common and can cause cancer and other diseases. However, our understanding of the mutability in these genes remains rudimentary. Therefore, given previously known factors which are associated with high mutation rates, we analyzed how many genes encoding druggable kinases match (i) proximity to telomeres or (ii) high A+T content. We extracted this genomic information using the National Institute of Health Genome Data Viewer. First, among 129 druggable human kinase genes studied, 106 genes satisfied either factors (i) or (ii), resulting in an 82% match. Moreover, a similar 85% match rate was found in 73 genes encoding pro-inflammatory cytokines of multisystem inflammatory syndrome in children. Based on these promising matching rates, we further compared these two factors utilizing 20 de novo mutations of mice exposed to space-like ionizing radiation, in order to determine if these seemingly random mutations were similarly predictable with this strategy. However, only 10 of these 20 murine genetic loci met (i) or (ii), leading to only a 50% match. When compared with the mechanisms of top-selling FDA approved drugs, this data suggests that matching rate analysis on druggable targets is feasible to systematically prioritize the relative mutability-and therefore therapeutic potential-of the novel candidates.

Abstract 5011: Targeting kinome reprogramming in ESR1 fusion-driven breast cancer

Abstract Background: Transcriptionally active ESR1 gene fusions (ESR1-TAF) are a potent cause of estrogen receptor alpha-positive (ERα+) breast cancer endocrine therapy (ET) resistance. These ESR1-TAF are gain-of-function mutations, exhibiting estrogen-independent cell growth, motility and ET resistance. They are not directly druggable because the ERα C-terminal ligand binding domain (LBD) encoding sequence is replaced with a translocated in-frame partner gene sequence. Herein we utilized proteomic approaches to develop novel targeted therapies against ESR1-TAF driven tumorigenesis. Methods: ESR1 fusion cDNA constructs were expressed in ERα+ breast cancer cell lines (T47D and MCF7). Cell growth was assayed by an Alamar blue assay. A mass spectrometry (MS)-based Kinase Inhibitor Pulldown Assay (KIPA) was employed to identify druggable kinases that are commonly upregulated by diverse ESR1-TAFs. A panel of 22 ERα+ patient-derived xenograft (PDX) models were profiled using proteomics and phosphoproteomics to identify models with sensitivity to RET kinase inhibition. Results: KIPA detected an increased abundance of a receptor tyrosine kinase, RET, in T47D cells expressing ESR1-TAFs in an estrogen-independent manner, compared to stable cell lines expressing transcriptionally inactive fusions as well as wild-type ERα protein. Interestingly, RET was also increased when constitutive activating ERα LBD point mutants, Y537S and D538G, were expressed in breast cancer cells. Inhibition of the RET kinase in vitro by repurposing pralsetinib, an FDA-approved RET inhibitor for advanced thyroid and non-small-cell lung cancers, demonstrated a significant reduction in the growth of cells expressing ESR1-TAFs and ERα LBD mutants. These data nominate RET kinase as a common therapeutic vulnerability for ESR1-TAF expressing breast cancers. Proteomic profiling of 22 biologically heterogenous ERα+ PDX tumors defined targetable pathways and predicted tumor subsets that were responsive to RET inhibition therapy. Organoids and xenografts from the pan-ET resistant WHIM18 PDX (that expresses the ESR1-YAP1 TAF) were inhibited by pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. These data provide key preclinical rationale for the consideration of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. Interestingly, the growth of WHIM37 PDX (that expresses ERα D538G) that had low level of RET and high level of GFRα-1, the co-receptor of RET, was also suppressed by pralsetinib. This data suggests that either RET or GFRα-1 is a predictive biomarker for RET inhibitor efficacy. Conclusions: Kinome analysis of ESR1 translocated breast tumors using KIPA followed by drug sensitivity studies nominated RET as a new therapeutic target for ET-resistant ERα+ breast cancer. Citation Format: Xuxu Gou, Beom-Jun Kim, Meenakshi Anurag, Jonathan T. Lei, Meggie N. Young, Matthew V. Holt, Diana Fandino, Craig T. Vollert, Purba Singh, Mohammad A. Alzubi, Anna Malovannaya, Lacey E. Dobrolecki, Michael T. Lewis, Shunqiang Li, Matthew J. Ellis, Charles E. Foulds. Targeting kinome reprogramming in ESR1 fusion-driven breast cancer. [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 5011.

Abstract 2449: p38β promotes cancer stemness and chemoresistance in hepatocellular carcinoma

Abstract Protein kinases control a variety of oncogenic functions through a phosphotransfer dependent signal transduction cascade in cancers where potent and selective inhibitors could be developed with favorable pharmaceutical properties. In hepatocellular carcinoma (HCC), increasing evidence suggested that cancer stemness is a driving force of therapy resistance which is tightly controlled by kinase-dependent signaling cascades. However, there are still limited druggable kinase targets that are developed to combat cancer stemness and therapy resistance for HCC treatment. To identify potential kinase targets which promote stemness, survival and therapy resistance in HCC, two datasets profiling kinase genes which are important for HCC survival and chemoresistance in HCC spheres were compared. p38 MAPK beta (p38β) was found to be a common top-ranked kinase important for these functions. Clinically, biopsies of human HCC showed high expression of total and activated forms of p38β, suggesting that p38β could be a potential therapeutic target in HCC. Functionally, we demonstrated that p38β promoted HCC tumor initiation, self-renewal and resistance towards chemotherapy by cisplatin. Using tandem affinity purification coupled with mass spectrometry, we identified BiP, which is a chaperone protein, as a novel binding partner of p38β. Subsequent mechanistic validation and mass spectrometry analysis showed that p38β directly phosphorylates BiP at a previously unrecognized phosphorylation site. Rescue experiments with BiP mutation and inhibition abrogated the stemness and chemoresistant properties observed in p38β overexpressing HCC cells. Taken together, we identified and characterized p38β and its direct phosphorylation target BiP as critical regulators for promoting cancer stemness and chemoresistance in HCC. Citation Format: Ianto Bosheng Huang, Yunong Xie, Tin Lok Wong, Terence Kin Wah Lee, Jing-Ping Yun, Stephanie Kwai Yee Ma, Man Tong. p38β promotes cancer stemness and chemoresistance in hepatocellular carcinoma [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 2449.

Development and Utility of a PAK1-Selective Degrader.

Overexpression of PAK1, a druggable kinase, is common in several malignancies, and inhibition of PAK1 by small molecules has been shown to impede the growth and survival of such cells. Potent inhibitors of PAKs 1-3 have been described, but clinical development has been hindered by recent findings that PAK2 function is required for normal cardiovascular function in adult mice. A unique allosteric PAK1-selective inhibitor, NVS-PAK1-1, provides a potential path forward, but has modest potency. Here, we report the development of BJG-05-039, a PAK1-selective degrader consisting of NVS-PAK1-1 conjugated to lenalidomide, a recruiter of the E3 ubiquitin ligase substrate adaptor Cereblon. BJG-05-039 induced selective degradation of PAK1 and displayed enhanced anti-proliferative effects relative to its parent compound in PAK1-dependent, but not PAK2-dependent, cell lines. Our findings suggest that selective PAK1 degradation may confer more potent pharmacological effects compared with catalytic inhibition and highlight the potential advantages of PAK1-targeted degradation.

Integrated proteomics and phosphoproteomics revealed druggable kinases in neoadjuvant chemotherapy resistant tongue cancer.

Tongue squamous cell carcinoma is an aggressive oral cancer with a high incidence of metastasis and poor prognosis. Most of the oral cavity cancer patients present in clinics with locally advanced unresectable tumors. Neoadjuvant treatment is beneficial for these individuals as it reduces the tumor size aiding complete resection. However, patients develop therapy resistance to the drug regimen. In this study, we explored the differential expression of proteins and altered phosphorylation in the neoadjuvant chemotherapy resistant tongue cancer patients. We integrated the proteomic and phosphoproteomic profiles of resistant (n = 4) and sensitive cohorts (n = 4) and demonstrated the differential expression and phosphorylation of proteins in the primary tissue of the respective subject groups. We observed differential and extensive phosphorylation of keratins such as KRT10 and KRT1 between the two cohorts. Furthermore, our study revealed a kinase signature associated with neoadjuvant chemotherapy resistance. Kinases such as MAPK1, AKT1, and MAPK3 are predicted to regulate the resistance in non-responders. Pathway analysis showed enrichment of Rho GTPase signaling and hyperphosphosphorylation of proteins involved in cell motility, invasion, and drug resistance. Targeting the kinases could help with the clinical management of neoadjuvant chemotherapy-resistant tongue cancer.

Targeting kinome reprogramming in ESR1 fusion-driven metastatic breast cancer.

1085 Background: Genomic analysis has recently identified multiple ESR1 gene translocations in estrogen receptor-alpha positive (ERα+) metastatic breast cancer (MBC) that encode chimeric proteins whereby the ESR1 ligand binding domain is replaced by C-terminal sequences from many different gene partners. Transcriptionally active ESR1 fusions promoted hormone-independent cell growth, motility and resistance to endocrine therapy. The diversity of partner genes creates a considerable diagnostic challenge and no targeted treatments exist for ESR1 translocated tumors. Thus, we have established a transcriptional signature to diagnose the presence of an active ESR1 fusion (PMID: 34711608) and developed novel targeted therapies against ESR1 fusion-driven biology. Methods: Fifteen ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines by lentiviral transduction. Cell growth was assayed by Alamar blue assay. A mass spectrometry (MS)-based Kinase Inhibitor Pulldown Assay (KIPA) and tandem mass tag-based proteomics were performed to identify ESR1 fusion-driven druggable kinases for subsequent pharmacological inhibition. Results: KIPA profiling demonstrated an increase of multiple receptor tyrosine kinases including RET in T47D cells expressing active ESR1 fusions. Inhibition of RET by repurposing an FDA-approved drug significantly suppressed ESR1 fusion-driven cell growth in vitro, suggesting that despite marked diversity in the 3’ partners, common kinase activities were elevated and targetable. Proteogenomic profiling, including whole exome sequencing, RNA sequencing, and MS-based proteomics and phosphoproteomics were further performed on 22 ER+ patient-derived xenograft (PDX) tumors, which demonstrated different degrees of estradiol dependence. These integrated “omic” profiles defined targetable genes/pathways and predict tumor subsets that could be responsive to kinase inhibition therapy from this biologically heterogeneous panel of PDX tumors. WHIM18, a PDX naturally harboring the ESR1-YAP1 fusion showed elevated level of RET and CDK4/6 pathways. The tumor volumes were significantly reduced by the RET inhibitor. CDK4/6 inhibitor treatment showed similar tumor reductions to RET inhibition. Interestingly, WHIM9 PDX that expressed wild-type ESR1 conferred a comparable kinome profile to WHIM18. The tumor growth was significantly suppressed by RET or CDK4/6 inhibition. Therefore, pharmacological experiments validated proteogenomics-predicted drug response in two tested ER+ PDX models. Conclusions: Proteogenomics characterization of PDX tumors can drive clinical trial hypotheses. Here, we reveal therapeutic kinase vulnerabilities in ESR1 fusion-driven tumors as exemplified by RET inhibition, which will lay the framework for future clinical trials.

Evaluation of the Preclinical Efficacy of Lurbinectedin in Malignant Pleural Mesothelioma.

Simple SummaryThe marine drug lurbinectedin revealed an unprecedented efficacy against patient-derived malignant pleural mesothelioma cells, regardless of the histological type and the BAP1 mutation status. By inducing strong DNA damages, it dramatically arrested cell cycle progression and induced apoptosis. These results may be translated into the use of lurbinectedin as an effective agent for malignant pleural mesothelioma patients.Background: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer generally diagnosed at an advanced stage and characterized by a poor prognosis. The absence of alterations in druggable kinases, together with an immune-suppressive tumor microenvironment, limits the use of molecular targeted therapies, making the treatment of MPM particularly challenging. Here we investigated the in vitro susceptibility of MPM to lurbinectedin (PM01183), a marine-derived drug that recently received accelerated approval by the FDA for the treatment of patients with metastatic small cell lung cancer with disease progression on or after platinum-based chemotherapy. Methods: A panel of primary MPM cultures, resembling the three major MPM histological subtypes (epithelioid, sarcomatoid, and biphasic), was characterized in terms of BAP1 status and histological markers. Subsequently, we explored the effects of lurbinectedin at nanomolar concentration on cell cycle, cell viability, DNA damage, genotoxic stress response, and proliferation. Results: Stabilized MPM cultures exhibited high sensitivity to lurbinectedin independently from the BAP1 mutational status and histological classification. Specifically, we observed that lurbinectedin rapidly promoted a cell cycle arrest in the S-phase and the activation of the DNA damage response, two conditions that invariably resulted in an irreversible DNA fragmentation, together with strong apoptotic cell death. Moreover, the analysis of long-term treatment indicated that lurbinectedin severely impacts MPM transforming abilities in vitro. Conclusion: Overall, our data provide evidence that lurbinectedin exerts a potent antitumoral activity on primary MPM cells, independently from both the histological subtype and BAP1 alteration, suggesting its potential activity in the treatment of MPM patients.

Comprehensive Split TEV Based Protein-Protein Interaction Screening of the Hippo Signaling Pathway Identifies TAOK2 As Central Modulator

The conserved Hippo signaling pathway plays an important role in limiting tissue growth by controlling proliferation rates. The core of the pathway is composed of STK3/4 and LATS1/2 kinases, which restrict the activity of YAP1, the main downstream effector. Upstream, the pathway is controlled by polarity proteins, other kinases, receptors, and scaffolding proteins. We conducted a split TEV-based protein-protein interaction screen to evaluate physical interactions among major Hippo pathway components and potential upstream modulators. TAOK2 was identified as key modulator of Hippo signaling, binding to pathway core kinase cassette components. Especially, TAOK2 binds to and phosphorylates LATS1, regulating YAP1 phosphorylation states, YAP1 transcriptional activity, and the proliferation of HEK293 cells. In a subset of human cancers, reduced TAOK2 expression correlates with reduced patient survival time supporting TAOK2’s role as tumor suppressor gene. As TAOK2 is a principally druggable kinase, it represents an attractive pharmacological target to modulate growth.

P21-activated kinase 4 promotes the progression of esophageal squamous cell carcinoma by targeting LASP1.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors of the digestive tract in humans. Several studies have indicated that PAK4 is associated with the risk of ESCC and may be a potential druggable kinase for ESCC treatment. However, the underlying mechanism remains largely unknown. The aim of our study is to identify the functional role of PAK4 in ESCC. To determine the expression of PAK4 in ESCC, Western blot analysis and immunohistochemistry were performed, and the results showed that PAK4 is significantly upregulated in ESCC tissues and cell lines compared with normal controls and normal esophageal epithelial cell line. To further investigate the role of PAK4 in ESCC, cell viability assays, anchorage‐independent cell growth assays, wound healing assays, cellular invasion assays, in vivo xenograft mouse models, and metastasis assays were conducted, and the results showed that PAK4 can significantly facilitate ESCC proliferation and metastasis in vitro and in vivo. To determine the potential target of PAK4 in ESCC progression, a pull‐down assay was performed, and the results showed that LASP1 may be a potential target of PAK4. An immunoprecipitation assay and confocal microscopy analysis confirmed that PAK4 can bind to and colocalize with LASP1 in vitro and in cells. Notably, rescue experiments further illustrated the mechanistic network of PAK4/LASP1. Our research reveals the oncogenic roles of PAK4 in ESCC and preliminarily elucidates the mechanistic network of PAK4/LASP1 in ESCC.

Human Aurora kinase inhibitor Hesperadin reveals epistatic interaction between Plasmodium falciparum PfArk1 and PfNek1 kinases

Mitosis has been validated by numerous anti-cancer drugs as being a druggable process, and selective inhibition of parasite proliferation provides an obvious opportunity for therapeutic intervention against malaria. Mitosis is controlled through the interplay between several protein kinases and phosphatases. We show here that inhibitors of human mitotic kinases belonging to the Aurora family inhibit P. falciparum proliferation in vitro with various potencies, and that a genetic selection for mutant parasites resistant to one of the drugs, Hesperadin, identifies a resistance mechanism mediated by a member of a different kinase family, PfNek1 (PF3D7_1228300). Intriguingly, loss of PfNek1 catalytic activity provides protection against drug action. This points to an undescribed functional interaction between Ark and Nek kinases and shows that existing inhibitors can be used to validate additional essential and druggable kinase functions in the parasite.

Serum- and glucocorticoid- inducible kinase 2, SGK2, is a novel autophagy regulator and modulates platinum drugs response in cancer cells

For many tumor types chemotherapy still represents the therapy of choice and many standard treatments are based on the use of platinum (PT) drugs. However, de novo or acquired resistance to platinum is frequent and leads to disease progression. In Epithelial Ovarian Cancer (EOC) patients, PT-resistant recurrences are very common and improving the response to treatment still represents an unmet clinical need. To identify new modulators of PT-sensitivity, we performed a loss-of-function screening targeting 680 genes potentially involved in the response of EOC cells to platinum. We found that SGK2 (Serum-and Glucocorticoid-inducible kinase 2) plays a key role in PT-response. We show here that EOC cells relay on the induction of autophagy to escape PT-induced death and that SGK2 inhibition increases PT sensitivity inducing a block in the autophagy cascade due to the impairment of lysosomal acidification. Mechanistically we demonstrate that SGK2 controls autophagy in a kinase-dependent manner by binding and inhibiting the V-ATPase proton pump. Accordingly, SGK2 phosphorylates the subunit V1H (ATP6V1H) of V-ATPase and silencing or chemical inhibition of SGK2, affects the normal autophagic flux and sensitizes EOC cells to platinum. Hence, we identified a new pathway that links autophagy to the survival of cancer cells under platinum treatment in which the druggable kinase SGK2 plays a central role. Our data suggest that blocking autophagy via SGK2 inhibition could represent a novel therapeutic strategy to improve patients’ response to platinum.

Abstract 5677: Recurrent transcriptionally active ESR1 fusions render therapeutic vulnerabilities to kinase inhibition in advanced breast cancer

Abstract Background: We recently reported two ESR1 fusions (ESR1-YAP1 and ESR1-PCDH11X) that drive endocrine therapy (ET) resistance and metastasis in estrogen receptor positive (ER+) metastatic breast cancer (MBC) (PMC6171747). Here we report additional ESR1 fusions with diverse C-terminal partner genes - ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1 and ESR1-ARID1B. Their functional characteristics and effects on kinase biology will be described. Methods: ESR1 fusions were identified by RNA-seq in MBC using ChimeraScan to detect fusion junction reads. ESR1 fusion cDNA constructs were expressed in ER+ breast cancer cell lines. An alamarBlue assay assessed cell proliferation. RNA-seq followed by mRNA-qPCR assessed the transcriptional properties. A scratch wound assay assessed cell motility. A Kinase Inhibitor Pulldown (KIP) mass spectrometry-based assay was conducted to examine ESR1 fusion-driven druggable kinases. Results: All fusions retained the first 6 exons (e6) of ESR1, fused in-frame to C-terminal sequences of diverse partner genes. In addition to ESR1-YAP1 and ESR1-PCDH11X fusions, ESR1-SOX9 and ESR1-ARNT2 drove fulvestrant-resistant growth. RNA-seq revealed fusion-specific transcriptional signatures indicating enrichment of estrogen responsive and epithelial-to-mesenchymal transition (EMT) pathways that were confirmed by mRNA-qPCR. Transcriptionally active ESR1 fusions also promoted hormone-independent cell motility. KIP profiling demonstrated an increase in protein abundance of multiple receptor tyrosine kinases including RET and insulin like growth factor 1 receptor (IGF1R) in T47D cells expressing active ESR1 fusion proteins, both of which have been previously implicated in driving ET and MBC. Both proteins were also elevated in a patient-derived xenograft naturally harboring the ESR1-YAP1 fusion. Combinatorial inhibition of RET and IGF1R significantly suppressed ESR1 fusion-driven cell growth in vitro. Neither transcriptional activation nor kinase upregulation was observed in other ESR1-e6 fusions. Lastly, we report recurrent examples of specific known active ESR1 fusions: ESR1-PCDH11X (3 examples) (TEMPUS unpublished data) and ESR1-ARNT2 (2 examples) (PMC6872491). Conclusion: A subset of ESR1-e6 fusions are active, drive ET resistance and metastasis/EMT in experimental models. When ESR1 is fused in-frame with another transcriptional regulator, activity is predictable. However, recurrent partners that have non-transcriptional roles (PCDH11X) suggests cDNA expression-based functional screens should be continued. A common pattern of kinase activation indicates that ESR1 fusion specific therapeutic strategies could be devised. Citation Format: Xuxu Gou, Jonathan Lei, Beom-Jun Kim, Meenakshi Anurag, Sinem Seker, Saif Rehman, Adrian V. Lee, Kevin White, Michael Caldwell, Jonathan Ball, Dan R. Robinson, Matthew J. Ellis. Recurrent transcriptionally active ESR1 fusions render therapeutic vulnerabilities to kinase inhibition in advanced breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5677.

Abstract B06: Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway

Abstract We performed a large-scale profiling of currently available kinase inhibitors (KI) to identify “off-targets” that could be repurposed/repositioned as the starting point for the rational development of novel and selective kinase inhibitors against underdrugged targets. We profiled 257 KI at a single dose against a panel of 365 wild-type kinases tested individually, for a total of about 100,000 in vitro drug/kinase data points. After unsupervised hierarchical clustering of this dataset based on percent inhibition values, we identified a well-resolved subcluster driven by 5 KI with limited polypharmacology and a clean kinase-inhibition profile, mostly targeting AGC group kinases. These inhibitors were the AKT inhibitors GSK-690693, AZD-5363, and AT-7867; the MSK inhibitor SB-747651-A; and the Rho kinases (ROCK) inhibitor GSK-269962A. Notably, the AGC and hippo kinases LATS1 and LATS2 were part of this subcluster and were both inhibited to significant extents by these 5 compounds. With the regenerative potential of a candidate LATS1/2 inhibitor in mind, we next developed several orthogonal biochemical and cellular assays to prioritize compounds that possessed the desired activity against LATS1/2 without the burden of cytotoxic “off-targets,” such as those resulting from strong AKT inhibition. Based on the results of these assays, we prioritized one of the initially identified kinase inhibitors as the starting point for an ongoing, rational structure-activity relationship (SAR) effort to develop novel, 1st-in-class, highly selective LATS1 and LATS2 inhibitors. We will present data showing how such novel LATS1/2 inhibitors efficiently block LATS1/2-dependent YAP phosphorylation, induce YAP nuclear translocation in a high cell-density setting, and promote the transcription of bona fide YAP target genes such as AMOTL2, CTGF, and CYR61. Finally, consistently with the activation of a YAP-dependent transcriptional program, small-molecule inhibitors of LATS1/2 kinases promoted proliferation/regeneration in an in vitro wound-healing assay. Altogether, we identified novel small molecules that can be used as chemical probes to acutely inactivate the Hippo signaling pathway and validated LATS1 and LATS2 kinases as novel druggable kinases whose inhibition could be exploited for several indications, spanning from regenerative medicine to cancer immunotherapy. Citation Format: Michele Ceribelli, Patrick Morris, Damien Duveau, Frances A. Tosto, Scott Hoyt, Craig J. Thomas. Development of selective LATS1/LATS2 inhibitors for the pharmacologic modulation of the Hippo signaling pathway [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B06.

Abstract A08: A new driver pathway in ovarian cancer stem cells

Abstract Ovarian cancer stem cells (OCSC) are believed to fuel tumor metastasis, recurrence, and chemoresistance. Therefore, understanding the molecular players involved in OCSC function represents an urgent need towards the design of innovative and more efficacious treatments of this neoplasm. Our molecular profiling of primary OCSC isolated from high-grade serous ovarian cancer patients revealed a series of novel genes and pathways specifically associated with this cell subpopulation. Among these, we initially focused on CD73, a cell-surface 5’-ectonucleotidase that catalyzes the production of extracellular adenosine. Our data revealed the new role of CD73 as a driver of OC stemness and a potential target for OCSC-eradicating treatments. More recently, we investigated the functional role in OCSC of the immunoglobulin-like cell adhesion molecule L1 (L1CAM), a transmembrane protein that we have previously implicated in OC malignancy (Zecchini et al., 2008). Loss- and gain-of-function studies were performed by manipulating the expression of L1CAM in OC cells. These experiments revealed the causal role of L1CAM not only in stemness-related properties such as anoikis resistance, clonogenic proliferation under low-attachment conditions and self-renewal, but also in OCSC-driven tumor initiation. Mechanistically, L1CAM appeared to act through the induction, activation, and nuclear translocation of STAT3, all events that occurred predominantly in the OCSC compartment rather than in the bulk tumor cell population. Indeed, the inhibition of STAT3 activity abolished L1-induced OC stemness. Of note, L1-dependent activation of STAT3 was not mediated by the canonical JAK pathway, but rather by the nonreceptor tyrosine kinase SRC. Accordingly, SRC inhibition prevented the induction of stemness-related features by L1CAM. Finally, to identify the molecular mechanisms that link L1CAM with SRC, we built on our previous findings that the L1CAM-related molecule NCAM activates FGFR1 signaling. This enabled us to find a crosstalk between L1CAM and FGFR1 which, in turn, accounts for the stimulation of SRC-mediated activation of STAT3 and, ultimately, OCSC function. Finally, we provide evidence that the inactivation of L1CAM enhances the sensitivity of OCSC to chemotherapeutics, implicating L1CAM as a potential target to overcome chemoresistance. In conclusion, our results uncovered a novel L1/FGFR1/SRC/STAT3 pathway that acts as a driver in OC stemness, shedding light on the pathophysiology of OCSC and opening new therapeutic perspectives for the eradication of such a devastating disease.

Abstract IA11: The use of GEM tumor models to identify effective therapies against KRAS mutant tumors

Abstract KRAS oncogenes are responsible for one fourth of all human tumors, including lung and pancreatic adenocarcinomas, two tumor types with some of the worse prognoses. Unfortunately, development of suitable therapies to treat these tumors has remained elusive for the last 35 years. Although KRAS remains an undruggable target (with the possible exception of the KRASG12C isoform), most of its downstream effectors are druggable kinases. Indeed, pharma and biotech companies have already developed dozens of selective inhibitors against these kinases. Yet none of them has been approved by the FDA, mostly due to unacceptable toxicities at antitumor effective doses. Hence, patients are still treated with old chemotherapy drugs. To address this important health issue, we have used a new generation of genetically engineered mouse (GEM) tumor models of KRas/Tp53 driven lung and pancreatic tumors to deconstruct oncogenic KRas signaling to identify those effectors whose systemic ablation/inactivation in tumor-bearing mice will result in therapeutic responses without inducing unacceptable toxicities. We have used this experimental strategy to interrogate the effect of ablating/inactivating each of the members of the MAPK cascade, including the Raf, Mek, and Erk kinases, as well as key effectors of the PI3K pathway, including PI3K p110alpha and mTOR. We have also evaluated additional downstream signaling elements such as the cell cycle Cdks. This systematic approach has revealed that most of the KRas signaling effectors are not suitable therapeutic targets due to either lack of therapeutic activity, such as Cdk2, Cdk6 A-Raf, or B-Raf, or to the induction of unacceptable toxicities such as the Mek1/2 and Erk1/2 kinases, PI3K p110alpha, mTOR, and Cdk1. Only Raf1 and Cdk4 turned out to be suitable therapeutic targets, based not only on their antitumor properties but also on the well-tolerated toxicities. Surprisingly, EGFR, an upstream effect of KRas, also has suitable therapeutic activity in pancreatic but not in lung tumors. We are now combining these targets to define more efficacious therapeutic strategies that could be eventually translated to the clinic. Moreover, we are using these experimental GEM tumor models to identify potential resistance mechanisms to these therapies. Citation Format: Mariano Barbacid. The use of GEM tumor models to identify effective therapies against KRAS mutant tumors [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr IA11.

Abstract IA01: Deconstructing K-RAS oncogenic signaling in lung and pancreatic tumors

Abstract The presence of K-RAS oncogenes in human tumors has been known for over 35 years. Indeed, extensive analysis of cancer genomes has revealed that they are present in at least a quarter of all solid tumors. Moreover, there is overwhelming evidence that they represent the initiating event in lung and pancreatic adenocarcinomas, two cancer types with some of the worse prognoses. Recent studies in genetically engineered mouse (GEM) tumor models have shown that inhibition of K-RAS oncogene expression results in rapid tumor regression. Yet in spite of recent progress, K-RAS oncogenes remain for the most part undruggable. Ironically, the main K-RAS downstream effectors are druggable kinases. Selective inhibitors against members of the MAPK and PI3K signaling pathways have been developed over the last two decades. Yet none of them has yielded suitable results in clinical trials to merit FDA approval against K-RAS mutant malignancies. In an effort to identify K-RAS vulnerabilities that may have been missed using biochemical or pharmacologic approaches, we decided a decade ago to deconstruct K-Ras oncogenic signaling in GEM tumor models by interrogating the effect of ablating or inactivating its downstream kinases during tumor initiation. In addition, we examined the consequences of systemically eliminating or inhibiting these targets during adult homeostasis, in order to evaluate the potential toxic effects that may result from tampering with the MAPK and PI3K signaling pathways. These studies revealed three classes of downstream targets: those whose ablation had no consequences on tumor development, those that prevented tumor development but were unacceptably toxic when ablated systemically, and a limited number (c-Raf and Cdk4) that prevented or thwarted tumor development without causing significant toxicities upon systemic elimination. In addition to these downstream targets, we were also able to identify the EGFR as a potential target in pancreatic but not in lung tumors. More recently, we have established a new generation of “therapeutic” GEM tumor models in which we can separate, both spatially and temporally, tumor development from target ablation/inactivation by using two independent recombinase systems. In these models, whereas K-Ras or K-Ras/TP53-driven tumor development is mediated by the Flp(o)/frt system, we use the inducible CreERT2/loxP system for systemic target ablation/inactivation. These “therapeutic” models have allowed us to explore the therapeutic potential of these previously validated targets in tumor-bearing mice. Our results have revealed that c-Raf ablation induces significant levels of tumor regression in K-Ras/TP53-driven lung tumors, a therapeutic effect that is further potentiated by the concomitant expression of a kinase dead isoform of Cdk4. In the corresponding GEM pancreatic tumor model, ablation or inactivation of c-Raf, Cdk4, or EGFR failed to prevent tumor regression, although tampering with Cdk4 or EGFR resulted in significant tumor delays. Importantly, concomitant ablation of c-Raf and EGFR in K-Ras/TP53-driven advanced pancreatic tumors resulted in complete tumor regression in half of the mice. Preliminary comparison of these “responder” tumors with those that did not respond to c-Raf/EGFR ablation is revealing some vulnerabilities that may provide additional targets against these advanced tumors. We hope that these results may serve to pave the way to define more efficacious therapeutic strategies against K-RAS mutant tumors that might eventually be translated to the clinic. Citation Format: Mariano Barbacid. Deconstructing K-RAS oncogenic signaling in lung and pancreatic tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA01.