Cdc2-like Kinase Research Articles

Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR-Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncover a selective dependency on RNA splicing factors whose loss preferentially enhances response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augments response to venetoclax in leukemia yet is completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition leads to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. Inhibition of splicing kinase families CLKs (CDC-like kinases) and DYRKs (dual-specificity tyrosine-regulated kinases) leads to aberrant splicing of key splicing and apoptotic factors that synergize with venetoclax, and overcomes resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments.

Straightforward Access to a New Class of Dual DYRK1A/CLK1 Inhibitors Possessing a Simple Dihydroquinoline Core.

The DYRK (Dual-specificity tyrosine phosphorylation-regulated kinase) family of protein kinases is involved in the pathogenesis of several neurodegenerative diseases. Among them, the DYRK1A protein kinase is thought to be implicated in Alzheimer's disease (AD) and Down syndrome, and as such, has emerged as an appealing therapeutic target. DYRKs are a subset of the CMGC (CDK, MAPKK, GSK3 and CLK) group of kinases. Within this group of kinases, the CDC2-like kinases (CLKs), such as CLK1, are closely related to DYRKs and have also sparked great interest as potential therapeutic targets for AD. Based on inhibitors previously described in the literature (namely TG003 and INDY), we report in this work a new class of dihydroquinolines exhibiting inhibitory activities in the nanomolar range on hDYRK1A and hCLK1. Moreover, there is overwhelming evidence that oxidative stress plays an important role in AD. Pleasingly, the most potent dual kinase inhibitor 1p exhibited antioxidant and radical scavenging properties. Finally, drug-likeness and molecular docking studies of this new class of DYRK1A/CLK1 inhibitors are also discussed in this article.

MSC-1186, a Highly Selective Pan-SRPK Inhibitor Based on an Exceptionally Decorated Benzimidazole-Pyrimidine Core

The highly conserved catalytic sites in protein kinases make it difficult to identify ATP competitive inhibitors with kinome-wide selectivity. Serendipitously, during a dedicated fragment campaign for the focal adhesion kinase (FAK), a scaffold that had lost its initial FAK affinity showed remarkable potency and selectivity for serine-arginine-protein kinases 1-3 (SRPK1-3). Non-conserved interactions with the uniquely structured hinge region of the SRPK family were the key drivers of the exclusive selectivity of the discovered fragment hit. Structure-guided medicinal chemistry efforts led to the SRPK inhibitor MSC-1186, which fulfills all hallmarks of a reversible chemical probe, including nanomolar cellular potency and excellent kinome-wide selectivity. The combination of MSC-1186 with CDC2-like kinase (CLK) inhibitors showed additive attenuation of SR-protein phosphorylation compared to the single agents. MSC-1186 and negative control (MSC-5360) are chemical probes available via the Structural Genomics Consortium chemical probe program (https://www.sgc-ffm.uni-frankfurt.de/).

A First-in-Human Phase I Study of CTX-712 in Patients with Advanced, Relapsed or Refractory Malignant Tumors - Hematologic Malignancies Dose Escalation Cohort

Background: Mutations of RNA splicing factors, including SF3B1, SRSF2, U2AF1, and ZRSR2, are implicated in the pathogenesis of various cancers, such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), most likely via abnormal RNA splicing. CTX-712 is a first-in-class, orally available, highly potent, and selective small-molecular inhibitor of CDC2-like kinase (CLK), a key regulator of the RNA splicing process. Preclinical data show that CTX-712 demonstrated anti-proliferative activity on various in vitro and in vivo models derived from not only solid tumors but also hematologic malignancies. Based on these results, a phase I study of CTX-712 was conducted to determine the recommended dose (RD) in solid tumors and hematologic malignancies. While therapeutic agents have been developed to target RNA splicing factor abnormalities, no such agents have been developed to be standard care until now. We report the interim data from the dose escalation cohort of relapsed or refractory AML and MDS, including preliminary efficacy results of CTX-712. Methods: The objectives of this cohort are to evaluate the maximum tolerated dose, dose- limiting toxicity (DLT), safety, pharmacokinetic/pharmacodynamic profiles, and preliminary efficacy of CTX-712 and to determine the RD for the next phase in patients with hematologic malignancies. A 3+3 design is used for finding the RD. An initial CTX-712 dose of 105 mg twice a week was used based on the safety information of the preceding solid tumor dose escalation cohort. Results: As of April 28, 2022, a total of 6 patients (5 AML and 1 MDS) were enrolled in the initial 105 mg twice-a-week dosing cohort. Three of the 6 patients were considered non-evaluable for DLT analysis (2 patients received less than 75% of the dose in cycle 1 due to tumor lysis syndrome, 1 patient discontinued treatment due to disease progression before completing cycle 1). A DLT of Grade 4 pneumonia was observed in 1 of 3 DLT-evaluable patients. Based on these safety profiles, the safety monitoring committee recommended to suspend the 105 mg twice-a-week dosing cohort and to initiate a 70 mg twice-a-week dosing cohort. Among the safety analysis population (N=6), with a median treatment duration of 114 days (Range 15-609 days), the common any-grade adverse events (AEs) (≥30%) were nausea (100%), diarrhea, hypotension (66.7%), vomiting, blood creatinine increased, pyrexia, insomnia (50.0%), fatigue, malaise, amylase increased, tumour lysis syndrome, dysgeusia, nail discoloration, dry skin, rash, and oedema (33.3%). The most common Grade 3 or higher AE was tumour lysis syndrome (33.3%). In the AML patients (N=5), complete remission (CR) was observed in two patients (40.0%), and CR with incomplete hematologic recovery (CRi) was observed in one patient (20.0%). The median time to remission is 56 days (Range 29-57 days), and the median duration of response is 56 days (Range 49+-532 days). In the MDS patient (N=1), CR was observed. Pharmacokinetic/pharmacodynamic profiles of the hematologic malignancies cohort were comparable with those of the solid tumor cohort in the same dosage. Conclusions: Assessment of CTX-712 safety profile for hematologic malignancies is ongoing. CTX-712 showed preliminary anti-tumor efficacy for both AML and MDS. Clinical trial information: JapicCTI-184188

CNSC-06. TEMOZOLOMIDE-INDUCED GUANINE MUTATIONS CREATE EXPLOITABLE VULNERABILITIES OF GUANINE-RICH DNA AND RNA REGIONS IN DRUG-RESISTANT GLIOMAS

Abstract Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive braincancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-lineinterventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to targetguanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We reportthat TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulatedalternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizingdrug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Lastly, we show that the G4 and RNA binding proteinEWSR1, member of the FUS, EWSR1, and TAF15 family, aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

Role of protein phosphorylation in cell signaling, disease, and the intervention therapy.

Protein phosphorylation is an important post-transcriptional modification involving an extremely wide range of intracellular signaling transduction pathways, making it an important therapeutic target for disease intervention. At present, numerous drugs targeting protein phosphorylation have been developed for the treatment of various diseases including malignant tumors, neurological diseases, infectious diseases, and immune diseases. In this review article, we analyzed 303 small-molecule protein phosphorylation kinase inhibitors (PKIs) registered and participated in clinical research obtained in a database named Protein Kinase Inhibitor Database (PKIDB), including 68 drugs approved by the Food and Drug Administration of the United States. Based on previous classifications of kinases, we divided these human protein phosphorylation kinases into eight groups and nearly 50 families, and delineated their main regulatory pathways, upstream and downstream targets. These groups include: protein kinase A, G, and C (AGC) and receptor guanylate cyclase (RGC) group, calmodulin-dependent protein kinase (CaMK) group, CMGC [Cyclin-dependent kinases (CDKs), Mitogen-activated protein kinases (MAPKs), Glycogen synthase kinases (GSKs), and Cdc2-like kinases (CLKs)] group, sterile (STE)-MAPKs group, tyrosine kinases (TK) group, tyrosine kinase-like (TKL) group, atypical group, and other groups. Different groups and families of inhibitors stimulate or inhibit others, forming an intricate molecular signaling regulatory network. This review takes newly developed new PKIs as breakthrough point, aiming to clarify the regulatory network and relationship of each pathway, as well as their roles in disease intervention, and provide a direction for future drug development.

An overview of cdc2-like kinase 1 (Clk1) inhibitors and their therapeutic indications.

Over the past decade, Clk1 has been identified as a promising target for the treatment of various diseases, in which deregulated alternative splicing plays a role. First small molecules targeting Clk1 are in clinical trials for the treatment of solid cancer, where variants of oncogenic proteins derived from alternative splicing promote tumor progression. Since many infectious pathogens hi-jack the host cell's splicing machinery to ensure efficient replication, further indications in this area are under investigation, such as Influenza A, HIV-1 virus, and Trypanosoma infections, and more will likely be discovered in the future. In addition, Clk1 was found to contribute to the progression of Alzheimer's disease through causing an imbalance of tau splicing products. Interestingly, homozygous Clk1 knockout mice showed a rather mild phenotype, opposed to what might be expected in view of the profound role of Clk1 in alternative splicing. A major drawback of most Clk1 inhibitors is their insufficient selectivity; in particular, Dyrk kinases and haspin were frequently identified as off-targets, besides the other Clk isoforms. Only few inhibitors were shown to be selective over Dyrk1A and haspin, whereas no Clk1 inhibitor so far achieved selectivity over the Clk4 isoform. In this review, we carefully compiled all Clk1 inhibitors from the scientific literature and summarized their structure-activity relationships (SAR). In addition, we critically discuss the available selectivity data and describe the inhibitor's efficacy in cellular models, if reported. Thus, we provide a comprehensive overview on the current state of Clk1 drug discovery and highlight the most promising chemotypes.

Novel prognostic model predicts overall survival in colon cancer based on RNA splicing regulation gene expression

Colon cancer is the third most common cancer and the second leading cause of cancer‐related death worldwide. Dysregulated RNA splicing factors have been reported to be associated with tumorigenesis and development in colon cancer. In this study, we interrogated clinical and RNA expression data of colon cancer patients from The Cancer Genome Atlas (TCGA) dataset and the Gene Expression Omnibus (GEO) database. Genes regulating RNA splicing correlated with survival in colon cancer were identified and a risk score model was constructed using Cox regression analyses. In the risk model, RNA splicing factor peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PPARGC1) is correlated with a good survival outcome, whereas Cdc2‐like kinase 1(CLK1), CLK2, and A‐kinase anchor protein 8‐like (AKAP8L) with a bad survival outcome. The risk model has a good performance for clinical prognostic prediction both in the TCGA cohort and the other two validation cohorts. In the tumor microenvironment (TME) analysis, the immune score was higher in the low‐risk group, and TME‐related pathway gene expression was also higher in low‐risk group. We further verified the mRNA and protein expression levels of these four genes in the adjacent nontumor, tumor, and liver metastasis tissues of colon cancer patients, which were consistent with bioinformatics analysis. In addition, knockdown of AKAP8L can suppress the proliferation and migration of colon cancer cells. Animal studies have also shown that AKAP8L knockdown can inhibit tumor growth in colon cancer in vivo. We established a prognostic risk model for colon cancer based on genes related to RNA splicing regulation and uncovered the role of AKAP8L in promoting colon cancer progression.

CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation

Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy and heart failure. Knockdown of Clk4 leads to pathological cardiomyocyte hypertrophy, while overexpression of Clk4 confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific Clk4-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by Clk4 knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific Clk4 deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.

Temozolomide-induced guanine mutations create exploitable vulnerabilities of guanine-rich DNA and RNA regions in drug-resistant gliomas.

Temozolomide (TMZ) is a chemotherapeutic agent that has been the first-line standard of care for the aggressive brain cancer glioblastoma (GBM) since 2005. Although initially beneficial, TMZ resistance is universal and second-line interventions are an unmet clinical need. Here, we took advantage of the known mechanism of action of TMZ to target guanines (G) and investigated G-rich G-quadruplex (G4) and splice site changes that occur upon TMZ resistance. We report that TMZ-resistant GBM has guanine mutations that disrupt the G-rich DNA G4s and splice sites that lead to deregulated alternative splicing. These alterations create vulnerabilities, which are selectively targeted by either the G4-stabilizing drug TMPyP4 or a novel splicing kinase inhibitor of cdc2-like kinase. Last, we show that the G4 and RNA binding protein EWSR1 aggregates in the cytoplasm in TMZ-resistant GBM cells and patient samples. Together, our findings provide insight into targetable vulnerabilities of TMZ-resistant GBM and present cytoplasmic EWSR1 as a putative biomarker.

Abstract 5389: Exploring potential predictive biomarkers for the responsiveness to CTX-712 by in vivo drug sensitivity analysis on PDX models

Abstract Cancer’s remarkable diversity had been understood through a logical framework of six key hallmarks. Additionally, several equally prevalent hallmarks have been identified in recent analyses of cellular phenotypes as the stress phenotypes of cancers. A novel hallmark of cancer RNA deregulation has been newly proposed as another hallmark of cancer within this group of stress phenotypes. However, the development of cancer therapies targeting RNA deregulation has only just begun. Indeed, small molecules that attack the RNA maturation processes and produce aberrant RNA are currently under development. We developed orally available, selective, and a first-in-class pan- CDC-like kinase (CLK) inhibitor, CTX-712, currently in phase 1 clinical trials for advanced, relapsed or refractory malignant cancers. CTX-712 dephosphorylates serine and arginine-rich proteins and induces primarily skipped exon type of splicing changes, resulting in the generation of RNA deregulation stress. In multiple preclinical models, excessive RNA deregulation stress caused cancer cell death and tumor growth inhibition. To explore the patient selection biomarkers for CTX-712, in vivo efficacy survey analysis has been conducted on breast, ovarian, colorectal, pancreatic, gastric and lung cancer models from patient derived xenograft (PDX) library with a detailed clinical information. Besides the efficacy investigation, the preclinical pharmacokinetics (PK) and pharmacodynamics (PD) analysis on tested PDX models were also performed to confirm plasma/tumor PK and PD (target CLK inhibition in xenografted tumors). Importantly, tested PDX models could be divided to sensitive (T/C on the most sensitive model = -71.1) and insensitive (T/C on the least sensitive model = 67.7), and the correlated tendency was observed between the efficacy and the inhibition magnitude of intratumor PD marker, production of exon skipped aberrant mRNA. To clarify the differences of sensitivity among tested PDXs will lead us to identity patient selection biomarkers of CTX-712. Therefore, whole transcriptome RNA sequencing, exome sequencing, and other approaches have been applied to reveal the differences of sensitivity among tested PDXs. Through this study on multiple PDXs, integration of drug responsiveness, and genomic/transcriptomic profiling may provide prediction markers for treatment responsiveness of CTX-712. Citation Format: Daisuke Morishita, Shigehiro Yagishita, Akio Mizutani, Hiroko Yamakawa, Shunsuke Ebara, Akinobu Hamada. Exploring potential predictive biomarkers for the responsiveness to CTX-712 by in vivo drug sensitivity analysis on PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5389.

Abstract 3928: The pan-CLK/DYRK inhibitor cirtuvivint selectively disrupts alternative splicing and has broad anti-tumor activity in preclinical models

Abstract Dysregulated alternative pre-mRNA splicing (AS) is commonly associated with human malignancies, producing pathological proteomes that underlie disease initiation, progression, and therapeutic resistance. The CDC2-like kinases (CLKs) and dual-specificity tyrosine-regulated kinases (DYRKs) are thought to govern AS efficiency and specificity by directly phosphorylating splicing factors that influence pre-mRNA splice junction selection. Cirtuvivint (SM08502) is a first-in-class small molecule ATP-competitive inhibitor of CLK/DYRK kinases. To evaluate the contribution of these kinases to AS profiles, high-depth RNAseq analysis was performed across 39 tumor cell lines (representing 9 lineages) and compared to 6 non-cancerous cell lines after treatment with cirtuvivint. Both baseline and drug-induced changes in AS events (ASEs) were measured using a multivariate analysis of transcript splicing (rMATS). Pan-CLK/DYRK inhibition was found to affect a minority of baseline ASEs, leaving the majority of spliceosome activity intact in all samples; however, the magnitude and quantity of detected drug-induced alterations were larger in tumor compared to non-cancerous cell lines. The majority of ASEs resulting from pan-CLK/DYRK inhibition were enriched for exon skipping events and were tumor type-specific. Moreover, drug-induced ASEs were significantly associated with disease-promoting biology across lineage and oncogenic driver contexts, including perturbed splicing of the AR-V7 variant in treatment-resistant prostate cancer and MDM2 in p53 wild-type cancers.To assess the breadth and depth of CLK/DYRK dependencies in human cancers, the effects of pan-CLK/DYRK inhibition on growth and survival of 154 cancer cell line models, 46 PDX models, and 43 CDX models were tested. EC50s in cell viability assays ranged from 0.014 to 0.73 μM in culture with strong effects observed in subsets of cell lines across all lineages tested. Multiomic data integration revealed sensitivity to cirtuvivint was associated with alterations in splicing genes. In vivo, tumor growth inhibition studies testing cirtuvivint at exposures ~2X below the MTD resulted in significant disease control (≥50% TGI) in 38/46 PDX and 38/43 CDX models. These observations indicate broad cancer relevance, at least in the preclinical setting, and expose vulnerabilities to CLK-DYRK-regulated splicing that can potentially be therapeutically addressed with pan CLK/DYRK inhibitors. Citation Format: Elizabeth A. McMillan, Carine Bossard, Emily Creger, Carsten Merkwirth, Raffaella Pippa, Matthew Jarvis, Krishna Sriram, Melinda Pedraza, Maureen Ibanez, Deepti Bhat, Carolyn Lai, Josh Stewart, Brian Eastman, Chi-Ching Mak, Michael A. White, Darrin Beaupre. The pan-CLK/DYRK inhibitor cirtuvivint selectively disrupts alternative splicing and has broad anti-tumor activity in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3928.

Abstract 165: Characterizing CLK3 in the colonic epithelium

Abstract The purpose of this study is to determine the function of CLK3 in the colonic epithelium and colon cancer. The intestinal epithelium undergoes constant renewal and is tightly regulated. Disruption of its homeostasis can result in the development of colorectal cancer (CRC). Glycogen synthase kinase 3 (GSK3) is a kinase that plays a central role in multiple signaling pathways including cellular proliferation. Our lab previously performed an siRNA screen to investigate hundreds of kinases to identify candidates which could interact with GSK3. Among the over 500 kinases that were tested, a protein kinase called CDC-like kinase 3 (CLK3) was identified as the strongest interaction with GSK3. We found that inhibition of GSK3 and CLK3 synergized to kill cancer cells. This led us to focus on CLK3 as a potential therapeutic target kinase in colon cancer. CLK3 is part of a four-member family of CDC-like kinases involved in pre-mRNA splicing. They are dual-specificity kinases, meaning they can target both tyrosine and serine/threonine substrates. CLK3 is also a poor prognostic marker in colon cancer, with high expression of CLK3 leading to poor outcome of disease. In human colonic epithelial cells, we show that loss of CLK3 dampens Wnt response. This data, along with the strong genetic interaction with GSK3, suggests a role of CLK3 in the Wnt pathway. Because Wnt signaling is higher in the stem cell region of the crypt, and CLK3 shows a role in Wnt activity in human colonic epithelial cells, we hypothesize that CLK3 regulates mRNA splicing to promote Wnt signaling and stemness in colonic progenitor cells and carcinogenesis. We are currently performing transcriptomics and phosphoproteomics in CLK3 CRISPR knockout cells to identify an mRNA-splicing and phosphorylation signature. We have found that the induction of Wnt-related pathway genes are lost in CLK3 knockout cells. Additionally, we are categorizing CLK3-dependent pre-mRNAs splicing events into functional groups controlling cell fate and validating in patient-derived normal and tumor organoid models. This work highlights CLK3 as a critical regulator of gut stem cells and potentially colorectal cancer stem cells. Citation Format: Carly Cabel, Curtis Thorne. Characterizing CLK3 in the colonic epithelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 165.

Abstract 2725: Molecular mechanisms of aberrant splicing in pediatric high grade gliomas

Abstract Pediatric brain cancer is the number one cause of disease-related death in children. While much of the somatic mutation variation in these tumors have been profiled, there have been few studies exploring their transcriptional splicing programs. Here, we characterize aberrant alternative splicing across pediatric brain tumors. We identified three splicing-based clusters that spanned across seven broad histological tumor classifications. Patients in clusters 1 and 2 had significantly lower overall survival (p < 0.001) than those in cluster 3. High-grade gliomas (HGGs) displayed the greatest splicing heterogeneity, spanning all three clusters. We focused on midline HGGs (n = 84), identifying 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. We also identified several splicing factor genes, including SRSF11, that were differentially expressed (p < 0.05), suggesting a potential tumor-specific novel mechanism of splicing dysregulation. Strikingly, 27% (5,157) of these splicing events resulted in either a gain or loss of a functional site in 3,294 genes: disulfide bonding (n = 391), localization signaling (n = 216), modifications, such as phosphorylation sites (n = 1,670), and other, such as ion-binding sites (n = 4,245). We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of the SR family of master splicing regulators, exhibit significantly increased exon 4 inclusion. This leads to a gain of two known phosphorylation sites in CLK1, increased protein expression, and hyper-phosphorylation of SR proteins, inducing cooperative binding of SR proteins to RNA. To assess the impact of this CLK1 event, we compared splicing in tumors with high exon 4 inclusion (n = 5) to those with low exon 4 inclusion (n = 5) and discovered 862 differential splicing events (DSE). Samples with high CLK1 exon 4 inclusion had significantly lower percent spliced in events, suggesting that increased activity of CLK1 resulting from the gain of the exon 4 promotes overall exon skipping. Additionally, more than 80% of the DSE (n = 695), including those in known brain tumor oncogenes such as CDC27 and E2H2, were recurrently mis-spliced in midline HGG tumors, suggesting that CLK1 splicing dysregulation leads to a global aberrant splicing phenotype in HGGs. Finally, we determined a subset of events significantly associated with prolonged survival, suggesting that stratification of patients based on splicing phenotypes can be utilized as a prognostic indicator. In summary, we demonstrate aberrant splicing in pediatric HGGs as an alternative mechanism driving tumorigenesis and future work will focus on urgently needed therapeutic targeting of these pathways toward our overall goal of improving survival outcomes in children with brain tumors. Citation Format: Ammar S. Naqvi, Brian Ennis, Run Jin, Karina L. Conkrite, Komal S. Rathi, Adam A. Kraya, Poonam Sonawane, Phillip B. Storm, Adam C. Resnick, Jo Lynne Rokita. Molecular mechanisms of aberrant splicing in pediatric high grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2725.

HGG-54. CLK1 aberrant splicing in pediatric high-grade gliomas disrupts key oncogenic transcriptional programs

Abstract While much of the somatic coding variation underlying the oncogenic transformation of pediatric high-grade gliomas (HGGs) has been profiled, transcriptional splicing programs of these tumors remain under-explored. Here, we characterize aberrant alternative splicing in pediatric midline HGGs (n = 84). We identified 19,275 recurrent and significant (20% change from control, P < 0.05, FDR < 0.05) aberrant splicing events in 8,587 genes compared to non-diseased brainstem controls. Of those, 27% (n = 5,157) resulted in either a gain or loss of a known protein functional site within 3,294 genes. We prioritized splice variants affecting targetable kinases and found that mRNAs encoding CDC-like kinase 1 (CLK1), a known modulator of master splicing regulators, exhibit significantly increased exon 4 inclusion in midline HGGs. This leads to a gain of two known phosphorylation sites in CLK1, increased CLK1 protein expression and hyper-phosphorylation of Serine-rich splicing factors. To assess the impact of this event, we performed differential splicing and expression analyses, comparing tumors with the highest (n= 5) and lowest (n = 5) exon 4 inclusion. We discovered 3,037 genes to be differentially up-regulated in high exon 4 inclusion tumors with an enrichment of cancer-related pathways, including DNA repair, mitotic spindle, myogenesis and EMT. We next integrated these gene signatures with protein-protein interaction networks of kinase and transcription factors and show that increased CLK1 exon 4 inclusion disrupts critical regulatory networks, such as those involving FOXM1, which is implicated in cell cycle and proliferation processes. In summary, we describe aberrant splicing in pediatric HGGs as an additional mechanism that could drive tumorigenesis. Future work will focus on molecular validation and therapeutic targeting of CLK1 in available HGG models. Characterizing tumor-specific splicing variation has the potential to open new therapeutic strategies and understand mechanisms of treatment resistance in children with central nervous system tumors.

A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors.

3080 Background: CTX-712 is a first in class, orally available, highly potent and selective small-molecular inhibitor of CDC2-like kinase (CLK), a key regulator of the RNA splicing process that plays a critical role in driving cell growth. CTX-712 demonstrated potent inhibition of proliferation in a variety of human tumor cell lines in vitro and elicited robust antitumor activity in vivo in multiple xenograft models. The objectives of this study are to determine the recommended dose (RD) by evaluating maximum tolerated dose (MTD) and dose limiting toxicity (DLT), safety, pharmacokinetics (PK) and pharmacodynamics (PD) profiles, and preliminary efficacy of CTX-712 in patients with solid tumors (ST) and hematologic malignancies (HM). Methods: This study consists of ST and HM dose escalation cohorts to identify MTD and ST dose expansion cohort to identify RD. The ST dose escalation cohort was initiated with accelerated titration and then switched to a 3+3 design (10, 20, 40, 70, 105, 140 and 175 mg/body twice a week in 28-day cycles). The initial dose of HM dose escalation cohort was decided from the safety information of ST dose escalation cohort. A 3+3 design was used in HM dose escalation cohort. Results: As of Dec. 31, 2021, 30 patients were enrolled (16 in the ST dose escalation cohort (10/20/40/70 mg [1], 105/175 mg [3], 140 mg [6]), 10 in the ST expansion cohort, and 4 in the HM dose escalation cohort). In the ST dose escalation cohorts, DLTs were observed in 2 patients (140 mg [platelet count decreased, hypokalemia], 175 mg [dehydration]) and MTD was determined to be 140 mg. Based on this safety information, the ST dose expansion cohort and the HM dose escalation cohort were initiated with the dose of 105 mg twice a week. Among all enrolled patients, the common any-grade Adverse Events (AEs) (≥30%) were nausea (97%), vomiting (63%), diarrhoea (63%), decreased appetite (57%), blood creatinine increased (40%), dysgeusia (37%), constipation (33%), pyrexia (33%) and white blood cell count decreased (30%). The most common Grade 3 or higher AEs were hypokalemia (10%), amylase increased and platelet count decreased (7%). In PK analysis, a dose-dependent increase in systemic exposure of CTX-712 was observed. PD response was assessed in RNA extracted from peripheral blood cells. Dose dependent increases of exon skipping in two marker RNAs were detected. Two Partial Responses and two Complete Responses were observed in ST and HM, respectively. Conclusions: CTX-712 demonstrated an acceptable safety profile with early signs of clinical antitumor activity, establishing the initial proof of concept of the CLK inhibitor. Observed DLTs included dehydration, platelet count decreased, and hypokalemia. Investigation is ongoing to determine RD. Clinical trial information: JapicCTI-184188.

Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Host-targeted self-attenuated influenza virus as a potential therapeutic influenza vaccine

Abstract Influenza is still one of leading causes of morbidity and mortality around the world, despite of annual influenza immunization in many countries. New strategies are urgently needed for developing effective vaccines and therapeutics against influenza viruses. In this study, we have constructed new recombinant influenza viruses, named host-targeted self-attenuated influenza viruses (SAIVs), which can express functional mammalian species-specific artificial microRNAs (amiRNAs). The expression of these amiRNAs can inhibit expression of some host factors critical for influenza replication, and therefore the resultant recombinant influenza viruses are replication restricted and attenuated in the host cells. One of these SAIVs, which can express an amiRNA that inhibits expression of the host cellular Cdc2-like kinase 1 (CLK1), was produced in embryonic chicken eggs and evaluated in a mouse model of influenza infection. It elicited robust antibody responses against influenza virus and demonstrated significantly protective efficacy against lethal infection with wild type influenza virus H1N1 PR8 after single dose of intranasal vaccination. Additionally, post-exposure treatment with this CLK1-targeted SAIV showed therapeutic effect against PR8 lethal-dose infection. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further developed to a therapeutic vaccine for prophylactic and therapeutic use against influenza. Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).

CLK2 upregulation and outcomes for males with colorectal adenocarcinoma.

159 Background: Colorectal adenocarcinoma (COAD) is a common cancer in gastrointestinal tract. CDC-like kinase (CLK) family, containing four characterized isoforms (CLK1-4), have been reported for their roles in precursor-mRNA splicing. However, studies on their roles in COAD are limited. Thus, this study was performed to explore the roles of CLK family members in COAD. Methods: RNA-sequencing FPKM data and corresponding clinical information of 41 normal tissues and 480 tumor tissues of COAD were obtained from The Cancer Genome Atlas (TCGA). Then expression differences of CLK family between the normal tissues and COAD cancer tissues were compared with Wilcoxon rank-sum test via ‘limma’ package. Overall survival (OS) of upregulated CLK family members was analyzed by Kaplan–Meier (K–M) method via ‘survminer’ package. Subgroup analyses of different genders were also conducted. The best discriminate cut-off point between the high and low expression groups was assessed by the receiver operating characteristic (ROC) curve and area under the curve (AUC) values for upregulated CLK family members via ‘pROC’ package to assess their diagnostic values. Results: The expression of CLK1 and CLK2 were upregulated in tumors compared with normal tissues (p < 0.001), while CLK3 were downregulated in tumor tissues (p = 0.02) and no statically significant difference was found regarding CLK4 (p = 0.25). Higher expression of CLK1 was not associated with a change of OS (HR: 1.24, 95%CI: 0.84-1.83, p = 0.279), while higher expression of CLK2 is associated with worse OS (HR: 2.03, 95%CI: 1.34-3.06, p = 0.001). Subgroup analysis found that higher expression of CLK2 was associated with worse OS (HR: 2.28, 95%CI 1.27-4.08, p = 0.006) in males, but not in females (HR: 1.72, 95%CI 0.95-3.10, P = 0.072). Further analysis of ROC curve shown that AUC of CLK1 and CLK2 were 0.748 (0.687-0.809) and 0.884 (0.840-0.928), respectively. Conclusions: CLK1 and CLK2 are upregulated in COAD tumor samples, and higher expression of CLK2 in male COAD patients is associated worse OS. This demonstrated the potential therapeutic value of CLK2 in male patients with COAD. ROC curve indicated the potential diagnostic value of CLK1 and CLK2.

The Identification of Prognostic and Metastatic Alternative Splicing in Skin Cutaneous Melanoma.

Skin cutaneous melanoma (SKCM) is a type of highly invasive cancer originated from melanocytes. It is reported that aberrant alternative splicing (AS) plays an important role in the neoplasia and metastasis of many types of cancer. Therefore, we investigated whether ASEs of pre-RNA have such an influence on the prognosis of SKCM and the related mechanism of ASEs in SKCM. The RNA-seq data and ASEs data for SKCM patients were obtained from the TCGA and TCGASpliceSeq database. The univariate Cox regression revealed 1265 overall survival-related splicing events (OS-SEs). Screened by Lasso regression, 4 OS-SEs were identified and used to construct an effective prediction model (AUC: .904), whose risk score was proved to be an independent prognostic factor. Furthermore, Kruskal–Wallis test and Mann–Whitney–Wilcoxon test showed that an aberrant splicing type of aminoacyl tRNA synthetase complex-interacting multifunctional protein 2 (AIMP2) regulated by CDC-like kinase 1 (CLK1) was associated with the metastasis and stage of SKCM. Besides, the overlapped signal pathway for AIMP2 was galactose metabolism identified by the co-expression analysis. External database validation also confirmed that AIMP2, CLK1, and the galactose metabolism were associated with the metastasis and stage of SKCM patients. ChIP-seq and ATAC-seq methods further confirmed the transcription regulation of CLK1, AIMP2, and other key genes, whose cellular expression was detected by Single Cell Sequencing. In conclusion, we proposed that CLK1-regulated AIMP2-78704-ES might play a critical role in the tumorigenesis and metastasis of SKCM via galactose metabolism. Besides, we established an effective model with MTMR14-63114-ES, URI1-48867-ES, BATF2-16724-AP, and MED22-88025-AP to predict the metastasis and prognosis of SKCM patients.