Cdc2-like Kinase Research Articles

2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases

Angiogenesis is a crucial process in the progression of various pathologies, like solid tumors, wet age-related macular degeneration, and chronic inflammation. Current anti-angiogenic treatments still have major drawbacks like limited efficacy in diseases that also rely on inflammation. Therefore, new anti-angiogenic approaches are sorely needed, and simultaneous inhibition of angiogenesis and inflammation is desirable. Here, we show that 2-desaza-annomontine (C81), a derivative of the plant alkaloid annomontine previously shown to inhibit endothelial inflammation, impedes angiogenesis by inhibiting CDC2-like kinases (CLKs) and WNT/β-catenin signaling. C81 reduced choroidal neovascularization in a laser-induced murine in vivo model, inhibited sprouting from vascular endothelial growth factor A (VEGF-A)-activated murine aortic rings ex vivo, and reduced angiogenesis-related activities of endothelial cells in multiple functional assays. This was largely phenocopied by CLK inhibitors and knockdowns, but not by inhibitors of the other known targets of C81. Mechanistically, CLK inhibition reduced VEGF receptor 2 (VEGFR2) mRNA and protein expression as well as downstream signaling. This was partly caused by a reduction of WNT/β-catenin pathway activity, as activating the pathway induced, while β-catenin knockdown impeded VEGFR2 expression. Surprisingly, alternative splicing of VEGFR2 was not detected. In summary, C81 and other CLK inhibitors could be promising compounds in the treatment of diseases that depend on angiogenesis and inflammation due to their impairment of both processes.Graphical abstract

Targeting the cGAS-STING Pathway Inhibits Peripheral T-cell Lymphoma Progression and Enhances the Chemotherapeutic Efficacy.

Peripheral T-cell lymphoma (PTCL) is a highly heterogeneous group of mature T-cell malignancies. The efficacy of current first-line treatment is dismal, and novel agents are urgently needed to improve patient outcomes. A close association between the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway and tumor promotion exists, revealing prospective therapeutic targets. This study, investigates the role of the cGAS-STING pathway and its underlying mechanisms in PTCL progression. Single-cell RNA sequencing showes that the cGAS-STING pathway is highly expressed and closely associated with PTCL proliferation. cGAS inhibition suppresses tumor growth and impaires DNA damage repair. Moreover, Cdc2-like kinase 1 (CLK1) is critical for residual tumor cell survival after treatment with cGAS inhibitors, and CLK1 suppression enhances sensitivity to cGAS inhibitors. Single-cell dynamic transcriptomic analysis indicates reduced proliferation-associated nascent RNAs as the underlying mechanism. In first-line therapy, chemotherapy-triggered DNA damage activates the cGAS-STING pathway, and cGAS inhibitors can synergize with chemotherapeutic agents to kill tumors. The cGAS-STING pathway is oncogenic in PTCL, whereas targeting cGAS suppresses tumor growth, and CLK1 may be a sensitivity indicator for cGAS inhibitors. These findings provide a theoretical foundation for optimizing therapeutic strategies for PTCL, especially in patients with relapsed/refractory disease.

Phase 1/2 Multicenter, Open-Label Study of CTX-712 in Patients with Relapsed/Refractory Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndromes

Background and Significance Dysregulation of alternative splicing, caused by abnormal expression or mutation of splicing factors, has been identified as an important contributor to the genesis and progression of cancer, and suggested as a novel target for anticancer treatment. CDC2-like kinases (CLK) represent a family of dual-specificity tyrosine and serine-threonine kinases that are involved in the regulation of splicing by phosphorylation of serine/arginine-rich splicing factors (SRSFs) which play a central role in exon recognition. CTX-712 is an orally available, potent, selective, and a first-in-class pan-CLK inhibitor (CLK 1-4), that inhibits phosphorylation of SRSFs resulting in significant antitumor activity in vitro and multiple xenograft models in vivo. CTX-712 formulated in capsules is currently being evaluated in an ongoing first-in-man study in Japan, evaluating tolerability and early efficacy in patients with solid tumors or hematologic malignancies (JapicCTI-184188). Preliminary data from the Japanese study were presented during ASCO 2022 and ASH 2022, and showed an acceptable safety profile (doses up to 175 mg (MTD 140 mg) twice a week in 28-day cycles, observed DLTs included dehydration, decreased platelets, hypokalemia, and pneumonia) with early signs of clinical antitumor activity (2 partial responses / 26 treated solid tumor patients, 5 complete remissions and complete remission with incomplete hematologic recovery/ 8 treated patients with AML or MDS), warranting further clinical investigations.. (J Clin Oncol (2022) 40(16_suppl):3080. Blood (2022) 140(Supplement 1):6211-2.). This abstract details a second study with CTX-712 that has just been initiated. Study Design and Methods A new film-coated tablet formulation will be evaluated in this phase 1/2 multicenter, open-label study of CTX-712 in patients with (mutated or wild type spliceosomes genes) relapsed/refractory AML, higher risk MDS (IPSS-R intermediate, high, or very high), or high marrow blast (≥5 %) MDS/MPN (including CMML). The phase 1 part of the study will be conducted at 4 US sites, and all sites are currently open for enrolment. The study allows patients with performance status 0, 1, or 2, adequate organ function, and up to four prior lines of therapy. Due to potential risk of interactions (in vitro studies suggest CYP3A4 as main metabolic pathway for CTX-712), concomitant use of strong CYP3A4 inhibitors is prohibited. Use of azole antifungals that are not strong CYP3A4 inhibitors (e.g., isavuconazole) is permitted. The phase 1 part of the study consists of dose-escalation (sequential standard 3 + 3 design), where cohorts of 3 (or 6) patients will receive ascending doses of CTX-712 once weekly in a 28-day cycle with continuous monitoring of emerging clinical PK, PD, safety, and efficacy data, to determine the recommended phase 2 dose (RP2D) based on the suggestion by the Safety Review Committee (SRC). The protocol defines five different dose levels (20-140 mg, once weekly) but also authorizes the SRC to suggest up to two interim dose levels or alternate schedules (e.g., dosing two times a week) if supported by emerging data. The suggested RP2D will be used in a confirmatory phase 1 expansion cohort where an additional 10 patients (total n = 16; 8 patients with AML and 8 patients with MDS) will be treated to gain further confidence in the selected dose level. The phase 2 part of the study will commence after the RP2D has been identified and confirmed and aims at evaluating therapeutic activity in R/R AML (Cohort 2a) or R/R HR-MDS (Cohort 2b), in addition to confirmation of the safety profile. Clinical trial registry number: NCT05732103

Interconnections between DDX41-Dependent Post-Transcriptional and GATA Factor-Instigated Transcriptional Mechanisms That Govern Hematopoiesis

Heterozygous DDX41 germline mutations occur in familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and acute erythroid leukemia (AEL). DDX41 encodes an RNA helicase that regulates RNA splicing, cGAS-Sting signaling in innate immunity, and maintains genome stability in erythropoiesis. We described the innovation of a facile genetic rescue system to compare RNA-regulatory activities of wild type and disease-associated DDX41 variants in engineered HoxB8-immortalized (hi) murine myeloid progenitor cells (hi-Ddx41+/+ and Ddx41+/- cells) (Kim et al., Leukemia 2023). We identified DDX41-regulated transcripts and membrane proteins as quantitative metrics of DDX41 activity that discriminate benign from pathogenic DDX41 alleles. As these transcripts and the cognate proteins were not known to be components of GATA2 genetic networks that govern hematopoietic stem/progenitor cell (HSPC) transitions, we hypothesized that DDX41 mechanisms in progenitors operate in parallel with or independent of GATA2 mechanisms. Considering the biological and pathological importance of DDX41 and many unanswered questions on how clinical variants impact DDX41 activity, we instituted studies to discover DDX41-regulated transcript isoforms. The datasets are being used to unveil how DDX41 controls cellular functions, how clinical variants derail mechanisms, and to establish interconnections or independence with GATA factor mechanisms. To develop a broader perspective, we extended the myeloid data by innovating a rescue system to analyze DDX41 structure/function in G1E-ER-GATA1 erythroblasts that express a conditionally active ER-GATA1 allele. We used CRISPR/Cas9 to generate Ddx41 +/- clonal lines that expressed ~50% lower levels of DDX41 protein. A comparable system is being generated in human HUDEP2 erythroid precursor cells. We compared DDX41 with two disease-associated germline variants: G173R and K331deletion. G1E-ER-GATA1- Ddx41 +/-cells were infected with retrovirus expressing GFP, or GFP and DDX41 (WT, G173R, or K331del), GFP + cells were sorted, and total RNA was analyzed by RNA-seq. Differential expression analysis revealed 138 Ddx41-regulated transcripts (P < 0.05), with 63 elevated and 75 decreased. Disease-associated variants failed to regulate these transcripts. To establish relationships between DDX41-regulated and GATA1-regulated transcripts in erythroblasts, we compared DDX41-regulated transcripts with our GATA1 transcriptomic data (Tanimura et al., EMBO Rep. 2016; Dev. Cell 2018; Liao et al., Cell Rep. 2020), which revealed 78 transcripts co-regulated by DDX41 and GATA1. The RNA analysis is being extended to the proteome. To investigate similarities between DDX41-dependent post-transcriptional and GATA1-dependent transcriptional mechanisms, we analyzed the genes encoding the 78 co-regulated transcripts using gene ontology and pathway analyses. The gene signatures included downregulation of apoptosis (p=0.0006). We are testing the hypothesis that DDX41 and GATA1 operate additively or synergistically to control levels of transcripts encoding apoptotic regulators in developing erythroblasts, thus establishing a balance between pro-apoptotic and anti-apoptotic factors. We compared myeloid and erythroid DDX41-regulated transcript datasets to identify common and cell type-specific DDX41-regulated transcripts. This analysis unveiled 11 transcripts that are DDX41-regulated in both systems. One transcript encodes CLK3, a member of Cdc2-like kinase family that phosphorylates splicing factors. DDX41 increased intron 3 retention in Clk3 mRNA in both systems. By quantifying exon-intron junctions, we demonstrated that DDX41 promoted intron 3 retention in hi- Ddx41 +/- cells 1.7-fold (P = 0.0002) and in G1E-ER-GATA1- Ddx41 +/- cells 3.3-fold (P = 0.0002). We are testing models to explain the functional significance of DDX41-regulated Clk3 intron retention and discovered DDX41 regulates intron retention at other loci. We are assessing how DDX41 controls alternative splicing globally in the erythroid and myeloid systems and elucidating the biological and pathological consequences of corrupting these mechanisms. In aggregate, these studies established a system to discriminate pathogenic from benign DDX41 variants, and mechanistic insights that provide the basis for new myeloid and erythroid paradigms.

Trial in Progress: A Phase 1b Single-Arm, Open-Label Study of Emavusertib (CA-4948) in Combination with Azacitidine and Venetoclax in Acute Myeloid Leukemia Patients in Complete Response with Measurable Residual Disease

Background Emavusertib is a novel potent oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4) with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Inhibition of these onco-proteins may induce remission thereby addressing a critical unmet need for novel therapies in acute myeloid leukemia (AML). Clinical studies with emavusertib monotherapy have demonstrated a significant reduction in AML blasts with clinical and molecular responses, including patients with relapsed or refractory AML, previously treated with an HMA and/or FLT3 inhibitors (Metzeler 2022). Azacitidine + venetoclax (aza+ven) has been approved in newly diagnosed, unfit patients with AML. In the VIALE-A study, composite complete response (CRc) (CR, CRh, or CRi) in the absence of measurable residual disease (MRD) of <1 residual blast/1000 leukocytes (MRD negative [MRD−]) resulted in longer duration of response (DOR), event-free survival, and overall survival (OS), and better HSCT outcome compared with patients who achieved CRc but were MRD+ (Pratz, 2022). In pre-clinical studies, emavusertib in combination with aza+ven demonstrated synergistic antileukemic effects in AML cell lines, including azacitidine- or venetoclax-resistant cell lines. Adding emavusertib to aza+ven in MRD+ patients at the time of CR may convert MRD status without adding significant toxicity and confirm that emavusertib can be safely added to aza+ven as a potential new regimen in front-line therapy. Study Design: This is a single-arm, open-label Phase 1b trial evaluating safety and tolerability, PK, and conversion of MRD status with emavusertib as an add-on agent to aza+ven in AML patients who achieved CR or CRh with MRD+ based on local testing (EU CT Number 2023-505828-58-00). The primary objective of the study is to determine a safe and tolerable dosing schedule for the triple combination. Secondary objectives include MRD conversion rate, DOR, OS, and pharmacokinetics. The study will enroll approximately 24 patients at 5 to 10 sites globally. Patients will have received azacitidine and venetoclax as first line therapy and achieved CR or CRh after 1-6 cycles of aza/ven. If MRD status remains positive, emavusertib will be added to the existing well tolerated aza+ven regimen. The starting emavusertib dose is 200 mg BID for 7 days per cycle of 28 days. If well tolerated, duration of emavusertib treatment will be extended to 14 and 21, respectively; no intra-patient change of emavusertib dosing duration is planned. The patients will continue triple treatment (emavusertib+aza+ven) until consent withdrawal, disease progression, intolerable toxicity, or not achieving MRD- within 6 cycles of triple treatment. In this Phase 1b trial, MRD can be evaluated by local testing of bone marrow. Key exclusion criteria include residual toxicities and significant comorbidities.

Takeaim Lymphoma: An Open-Label, Dose Escalation and Expansion Trial of Emavusertib (CA-4948) in Combination with Ibrutinib in Patients with Relapsed or Refractory Hematologic Malignancies

Background: Emavusertib (CA-4948) is a novel oral inhibitor of interleukin-1 receptor-associated kinase 4 (IRAK4), which is essential for toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling in B cell proliferation. Additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4) is also present in emavusertib. IRAK4 forms a Myddosome complex with MYD88 adaptor protein and drives overactivation ofnuclear factor-kappa B (NF-κB), causing inflammation and tumor growth. Emavusertib has been reported to be well tolerated and active as monotherapy in heavily pretreated patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) (Nowakowski 2020). Preclinical studies demonstrated that tumor resistance and survival via IRAK4 activation could be delayed or reversed. Emavusertib crossed the blood-brain barrier in a murine PDX model of PCNS lymphoma, blocked the activated IRAK4 pathway, and induced tumor response and prolonged survival (von Roemeling 2022). In combination with Bruton tyrosine kinase (BTK) inhibitors, emavusertib showed in vitro synergy and re-sensitization of resistant cell lines, overcoming BTK resistance (Guidetti 2023a and 2023b). In vivo synergy in B-cell NHL was also demonstrated. Adaptive chemotherapy treatment resistance through IRAK4 upregulation and activation was also described in other disease models (Melgar 2019, Li 2019). This abstract presents preliminary efficacy and safety data of emavusertib + ibrutinib in R/R NHL patients. Aim:Assessment of safety and clinical activity of emavusertib in combination with ibrutinib. Methods: This is an ongoing open-label trial (NCT03328078) of emavusertib as monotherapy and in combination with ibrutinib. Part A1 - completed; dose escalation of emavusertib as monotherapy; the recommended phase 2 dose (RP2D) is 300 mg BID with continuous oral dosing (Nowakowski 2020). Part A2 - completed; dose escalation of emavusertib in combination with ibrutinib at 420 and 560 mg QD (Joffe 2022). Part B - ongoing; an expansion cohort of PCNSL patients with resistance to prior BTK inhibitors for additional safety and efficacy related to exposure: emavusertib at 100 or 200 mg BID + ibrutinib at 560 mg QD in 28-Day cycles. Results:As of 19 July 2023, 18 heavily pretreated NHL (including five PCNSL) patients received emavusertib at 100, 200, or 300 mg BID in combination with full ibrutinib doses per label. Median age was 63.5 years (range 50-92). Median number of prior lines of anti-cancer therapies was 3 (range 1-10). Eleven of 18 patients failed prior BTK inhibitors. Median treatment duration was 95.5 days (21-587 days), suggesting acceptable safety and tolerability. No DLTs were observed at the 200 mg BID dose level, and two reversible DLTs (stomatitis and syncope) were observed at the 300 mg BID dose level. The preliminary efficacy data of 16 evaluable patients in combination with ibrutinib showed 5 CRs (2 MCL, 3 PCNSL) and 1 PR (1 CLL). All 3 PCNSL patients with CR were previously treated with BTK inhibitors. Conclusion: The combination ofemavusertib plus ibrutinib (ema+ibr) is well tolerated with an acceptable long-term safety profile and promising efficacy, showing several objective responses in heavily pretreated and/or BTK inhibitor resistant patients. Emavusertib may have the potential to overcome BTK inhibitor resistance and the combination of ema+ibr has the potential to show increased anti-cancer activity compared to ibrutinib monotherapy.

Trial in Progress: An Open-Label Expansion Trial Evaluating the Safety, PK/PD, and Clinical Activity of Emavusertib (CA-4948) + Ibrutinib in R/R Primary CNS Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) is a rare and aggressive form of NHL isolated to the central nervous system or vitreoretinal space. Despite high initial rates of responses to available therapies, most PCNLS relapse in two years, presenting a very poor prognosis. Combination with novel therapies may overcome resistance (Guidetti 2023a). Upregulation of the B-cell receptor (BCR/NF-κB) and toll-like receptor (TLR)/NF-κB) signaling axis have been identified as key pathways in the pathogenesis of PCNSL. MYD88 L265P, a widely expressed somatic mutation in more than 70% of PCNSL patients, activates NF-κB signaling through stimulation of BTK and IRAK4. Emavusertib (CA-4948) is a novel, potent oral inhibitor of interleukin 1 receptor-associated kinase 4 (IRAK4), with additional inhibitory activity against FMS-like tyrosine kinase 3 (FLT3) and CDC-like kinases (CLK1/2/4). Targeted inhibition of IRAK4 with emavusertib in PCNSL xenografts indicated sufficient blood brain barrier penetration of emavusertib, target inhibition, and antitumor activity (von Roemeling 2022). Further preclinical studies have demonstrated that emavusertib synergizes with ibrutinib and can re-sensitize and overcome resistance to BTK inhibitors (Guidetti 2023a and 2023b). Initial clinical data have reported responses in BTK-resistant PCNSL and good tolerance of the combination (Iqbal 2022; Grommes 2023). Study Design: This is an ongoing multi-center, open-label trial (NCT03328078) in adult patients with hematologic malignancies to evaluate the safety, PK, and anti-cancer activity of oral administration of emavusertib alone or in combination with ibrutinib. Part Bis an open trial expansion cohort with BTKi relapsed or refractory PCNSL. About 20 evaluable PCNSL patients will be enrolled globally. Patients will receive oral CA-4948 at 100 or 200 mg BID in combination with ibrutinib at 560 mg QD in 28-day cycles until disease progression or intolerable toxicity. The primary endpoint is expanded safety/tolerability; secondary endpoints include ORR, DOR, PFS, OS, and pharmacokinetics. Key inclusion criteria for this expansion cohort: pathologically confirmed diagnosis of PCNSL with evidence of disease progression, measurable disease, relapsed or refractory with no more than 3 lines of prior anti-PCNSL therapy (including BTKi), and absence of residual Grade ≥2 toxicity from prior therapy. Key exclusion criteria: intraocular PCNSL without brain lesion or CSF involvement or significant co-morbidity. Key clinical assessments include brain imaging, CSF- and ocular examinations.

CLASRP oncogene as a novel target for colorectal cancer

Clk4-associated serine/arginine-rich protein (CLASRP), an alternative splicing regulator, may be involved in the development and progression of cancer by regulating the activity of the CDC-like kinase (Clk) family. This study explored the biological function of CLASRP in colorectal cancer (CRC). The expression of CLASRP, which is associated with clinicopathological features, was analysed in CRC tissues and paired noncancer tissues by RT–PCR. The roles of CLASRP were investigated in CRC cells transfected with plasmids or shRNA through proliferation, migration and invasion assays in vitro and a xenograft model in vivo. Apoptosis was analysed using CLASRP-overexpressing CRC cells by western blotting. Clk inhibitors were used to perform functional research on CLASRP in CLASRP-overexpressing CRC cells. CLASRP was significantly upregulated in CRC cell lines, while high CLASRP expression was correlated with metastasis in CRC patients. Functionally, overexpression of CLASRP significantly promoted the proliferation, migration and invasion of CRC cells in vitro and tumour growth in vivo. Mechanistically, the proliferation, migration and invasion of CLASRP-overexpressing CRC cells were inhibited by Clk inhibitors, accompanied by low expression of CLASRP at the gene and protein levels. Clk inhibitors induced apoptosis of CLASRP-overexpressing CRC cells, resulting in direct blockade of cell growth. The expression levels of cleaved caspase 3 and cleaved caspase 8 were increased in CLASRP-overexpressing CRC cells treated with Clk inhibitors. CLASRP might serve as a promotional oncogene in CRC cells and be suppressed by Clk inhibitors through activation of caspase pathways.

CLK2 in GABAergic neurons is critical in regulating energy balance and anxiety-like behavior in a gender-specific fashion.

Cdc2-like kinase (CLK2) is a member of CLK kinases expressed in hypothalamic neurons and is activated in response to refeeding, leptin, or insulin. Diet-induced obesity and leptin receptor-deficient db/db mice lack CLK2 signal in the hypothalamic neurons. The neurotransmiter gamma-aminobutyric acid (GABA) is among the most prevalent in the central nervous system (CNS), particularly in the hypothalamus. Given the abundance of GABA-expressing neurons and their potential influence on regulating energy and behavioral homeostasis, we aimed to explore whether the deletion of CLK2 in GABAergic neurons alters energy homeostasis and behavioral and cognitive functions in both genders of mice lacking CLK2 in Vgat-expressing neurons (Vgat-Cre; Clk2loxP/loxP) on chow diet. We generated mice lacking Clk2 in Vgat-expressing neurons (Vgat-Cre; Clk2loxP/loxP) by mating Clk2loxP/loxP mice with Vgat-IRES-Cre transgenic mice and employed behavior, and physiological tests, and molecular approaches to investigate energy metabolism and behavior phenotype of both genders. We showed that deletion of CLK2 in GABAergic neurons increased adiposity and food intake in females. The mechanisms behind these effects were likely due, at least in part, to hypothalamic insulin resistance and upregulation of hypothalamic Npy and Agrp expression. Besides normal insulin and pyruvate sensitivity, Vgat-Cre; Clk2loxP/loxP females were glucose intolerant. Male Vgat-Cre; Clk2loxP/loxP mice showed an increased energy expenditure (EE). Risen EE may account for avoiding weight and fat mass gain in male Vgat-Cre; Clk2loxP/loxP mice. Vgat-Cre; Clk2loxP/loxP mice had no alteration in cognition or memory functions in both genders. Interestingly, deleting CLK2 in GABAergic neurons changed anxiety-like behavior only in females, not males. These findings suggest that CLK2 in GABAergic neurons is critical in regulating energy balance and anxiety-like behavior in a gender-specific fashion and could be a molecular therapeutic target for combating obesity associated with psychological disorders in females.

CMGC Kinases in Health and Cancer.

CMGC kinases, encompassing cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDC-like kinases (CLKs), play pivotal roles in cellular signaling pathways, including cell cycle regulation, proliferation, differentiation, apoptosis, and gene expression regulation. The dysregulation and aberrant activation of these kinases have been implicated in cancer development and progression, making them attractive therapeutic targets. In recent years, kinase inhibitors targeting CMGC kinases, such as CDK4/6 inhibitors and BRAF/MEK inhibitors, have demonstrated clinical success in treating specific cancer types. However, challenges remain, including resistance to kinase inhibitors, off-target effects, and the need for better patient stratification. This review provides a comprehensive overview of the importance of CMGC kinases in cancer biology, their involvement in cellular signaling pathways, protein-protein interactions, and the current state of kinase inhibitors targeting these kinases. Furthermore, we discuss the challenges and future perspectives in targeting CMGC kinases for cancer therapy, including potential strategies to overcome resistance, the development of more selective inhibitors, and novel therapeutic approaches, such as targeting protein-protein interactions, exploiting synthetic lethality, and the evolution of omics in the study of the human kinome. As our understanding of the molecular mechanisms and protein-protein interactions involving CMGC kinases expands, so too will the opportunities for the development of more selective and effective therapeutic strategies for cancer treatment.

CLK2 and CLK4 are regulators of DNA damage-induced NF-κB targeted by novel small molecule inhibitors

Transcription factor NF-κB potently activates anti-apoptotic genes, and its inactivation significantly reduces tumor cell survival following genotoxic stresses. We identified two structurally distinct lead compounds that selectively inhibit NF-κB activation by DNA double-strand breaks, but not by other stimuli, such as TNFα. Our compounds do not directly inhibit previously identified regulators of this pathway, most critically including IκB kinase (IKK), but inhibit signal transmission in-between ATM, PARP1, and IKKγ. Deconvolution strategies, including derivatization and invitro testing in multi-kinase panels, yielded shared targets, cdc-like kinase (CLK) 2 and 4, as essential regulators of DNA damage-induced IKK and NF-κB activity. Both leads sensitize to DNA damaging agents by increasing p53-induced apoptosis, thereby reducing cancer cell viability. We propose that our lead compounds and derivatives can be used in context of genotoxic therapy-induced or ongoing DNA damage to increase tumor cell apoptosis, which may be beneficial in cancer treatment.

Leucettinibs, a Class of DYRK/CLK Kinase Inhibitors Inspired by the Marine Sponge Natural Product Leucettamine B.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) recently attracted attention due to their central involvement in various pathologies. We here describe a family of DYRK/CLK inhibitors derived from Leucettines and the marine natural product Leucettamine B. Forty-five N2-functionalized 2-aminoimidazolin-4-ones bearing a fused [6 + 5]-heteroarylmethylene were synthesized. Benzothiazol-6-ylmethylene was selected as the most potent residue among 15 different heteroarylmethylenes. 186 N2-substituted 2-aminoimidazolin-4-ones bearing a benzothiazol-6-ylmethylene, collectively named Leucettinibs, were synthesized and extensively characterized. Subnanomolar IC50 (0.5-20 nM on DYRK1A) inhibitors were identified and one Leucettinib was modeled in DYRK1A and co-crystallized with CLK1 and the weaker inhibited off-target CSNK2A1. Kinase-inactive isomers of Leucettinibs (>3-10 μM on DYRK1A), named iso-Leucettinibs, were synthesized and characterized as suitable negative control compounds for functional experiments. Leucettinibs, but not iso-Leucettinibs, inhibit the phosphorylation of DYRK1A substrates in cells. Leucettinibs provide new research tools and potential leads for further optimization toward therapeutic drug candidates.

HGG-21. ABERRANT SPLICING OF CLK1 IN PEDIATRIC HIGH-GRADE GLIOMAS DISRUPTS KEY ONCOGENIC TRANSCRIPTIONAL PROGRAMS

Abstract While the majority of somatic DNA coding alterations in pediatric brain tumors have been profiled, studies exploring transcriptional mechanisms remain underexplored. We systematically characterized aberrant splicing in primary tumors of seven broad histologies of pediatric brain tumors. We obtained RNA-seq data (n = 792) from the Open Pediatric Brain Tumor Atlas and integrated rMATs splice event calls with UniProt Knowledgebase to quantify and prioritize differential alternative splicing events with proteomic potential. We identified three splicing-driven biological clusters and found pediatric high-grade gliomas (pHGGs) to be uniquely heterogeneous, spanning all three clusters. We also discovered that tumors with high splicing burden (SB) had significantly worse overall survival than tumors with low SB (p < 1x10-4). Furthermore, pHGGs (n = 154) displayed the greatest splicing heterogeneity with 20,620 exon splicing aberrations (pval/FDR < 0.05) in 3,294 genes that resulted in a gain or loss of known functional sites. We prioritized targetable kinases and found that CDC-like kinase 1 (CLK1), a known modulator of master splicing regulators, exhibited significantly increased exon 4 inclusion (p < 0.05, ΔPSI >= |.20|), and subsequent gain of two phosphorylation sites, in tumors compared to matched non-tumor tissue. Exon 4 inclusion was significantly correlated with increased CLK1 expression (r = 0.57, p = 7.8 x10-9), which we posit results in hyper-activity of CLK1 and aberrant splicing of its targets in these tumors. Furthermore, predicted CLK1 gene targets (n = 101) were enriched for cancer-related pathways, such as DNA repair (odds ratio > 2, p < 0.005). In summary, we describe aberrant splicing in pHGGs as an alternative mechanism that could drive tumorigenesis. Future work will focus on molecular validation and therapeutic targeting of CLK1 in available pHGG models, potentially opening new therapeutic strategies in children with brain tumors.

Alternative splicing (AS) regulation as a novel therapeutic target in soft-tissue sarcoma (STS).

11546 Background: There is an urgent clinical need to identify novel treatments for advanced STS, since all the clinical trials testing chemotherapy combinations have failed to improve survival compared to doxorubicin (dox) alone. AS deregulation has been associated with cancer initiation, progression and resistance to therapy. This deregulation can occur due to mutations or imbalanced expression or activity of splicing factors (SF). The alteration SF can alter thousands of pre-mRNAs, increasing cellular complexity and facilitating tumour progression. CDC-like kinases (CLK) that phosphorylate serine-arginine-rich SF (SRSFs), are key regulators of AS. SM09419 is an oral pan CLK/DYRK inhibitor that has been shown to inhibit SRSF phosphorylation, selectively modify spliceosome activity and decrease tumour growth by multiple mechanisms including the downregulation of Wnt/β-catenin signalling, a pathway that we reported to be important in sarcoma. Methods: A panel of 9 STS cell lines, including LMS (CP0024, IEC005, AA, SK-UT-1), LPS (93T449), synovial sarcoma (MCP037) and UPS (MCP016, MCP021, MCP025) were used to analyse the effect of SM09419 (provided by Biosplice) or PRI-724 (Wnt/β-catenin inhibitor) on cell viability (MTS assays). TOP flash β-catenin/TCF-responsive reporter assay was performed to evaluate the inhibition of the Wnt/β-catenin pathway in response to SM09419. In vivo experiments tested the combination of SM09419 (25mg/kg 5 days on and 2 off, for 21 days cycle) plus IP dox (5mg/kg once a week for 21 days cycle) in LMS (IEC005) and synovial sarcoma (FJD-SS-001) PDX models. Results: Our results showed that SM09419 had superior activity in STS cells compared to specific Wnt/β-catenin inhibitors with IC50 values starting at 29.8 nM in SK-UT-1 cell line, whereas PRI-724 IC50 values were clearly higher, starting from 5.18 µM in 93T449 cells. SM09419 treatment significantly inhibited the transcriptional activity of β-catenin by 40% in the MCP021 cells, demonstrating the regulatory activity of AS inhibitors on this pathway. I n vivo studies confirmed the higher efficacy of SM09419 in monotherapy compared to dox, in inhibiting tumour growth (p < 0.05), which was potentiated when combined with dox. The combination was increasingly active in synovial sarcoma with tumour regressions in 3 out of 4 mice, including one complete response. Treatment schedule was adapted to a single IP administration of dox (5mg/kg) and 5 administrations of SM09419 (25mg/kg), to improve the tolerability of the combination. Conclusions: The effects of pan CLK/DYRK inhibition on growth and survival of STS cancer cell lines and PDX models, indicate that can STS may be therapeutically addressed with these AS-modulators. The combination of SM09419 with dox was active in STS preclinical models known for their resilience toward treatment. Based on these results, the exploration of such combinations in clinical trials is warranted.

Host-targeted self-attenuated influenza virus as an effective nasal spray vaccine in aged population

Abstract The elderly are at high risk for influenza infection and its complication. However, current inactivated influenza vaccines are often modestly immunogenic and show low protective immunity in the elderly, due to immunosenescence. New approaches are urgently needed for developing effective vaccines against influenza for the elderly. In current 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 an aged mouse model of influenza infection. It elicited robust antibody and T cell 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. Our research finding provided a proof of concept that the new host targeted self-attenuated influenza virus can be further used as an effective vaccine against influenza in aged population. Supported by a research grant from National Institute of Allergy and Infectious Diseases (AI133207).

Abstract 3100: The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM)

Abstract Glioblastoma multiforme (GBM) is an aggressive stage IV brain cancer that is difficult to detect, typically inoperable, and associated with a poor prognosis. Challenges in targeting GBM arise from the involvement of various pro-survival pathways, being comprised of four different cellular states, and displaying heterogeneity between each tumor. Marketed FDA-approved drugs for GBM have demonstrated limited efficacy, toxicity issues, resistance, and poor BBB penetrance. Further probing into pathway breakdowns led to the discovery of the DYRKs (dual-specificity tyrosine-regulated kinase), CLKs (CDC-like kinases), PDGFRA/B (platelet-derived growth factor receptor), and PI3Kα (phosphoinositide 3-kinase) involvement and overexpression. The upregulation of these kinases are found to be prominently involved in GBM cell proliferation, survival, replication, aggression, metastasis, and resistance. Herein, we believe that the development of pleiotropic competitive inhibitors for the treatment of GBM could be a promising therapeutic option based on literature precedence and current data from research. DYR726, a water-soluble, pleiotropic inhibitor [IC50 PDGFRA/B/PI3Kα/CLk2/3DYRK1a/2/3 ( 53.0/48.0/6.2/15.13/137/4/38.6/11.2 nM)] has demonstrated in vitro cellular efficacy across various GBM cell lines. When treated in non-cancerous cell lines, DYR726 displayed an optimal therapeutic window, which is not observed in any glioma drugs currently in clinical trials. At 1µM concentrations, complete dissociation of the formation of neural stem cells and neurosphere growth in glioma cells are observed. Reduction in metastasis and proliferation are found when treated with DYR726 over three weeks. In vitro efficacy against current approved FDA treatment options and clinical candidates has been extensively tested across numerous GBM cell lines. When benched against one of the best PI3K kinase inhibitors in the clinic, Buparlisib, DYR726 was comparable, indicating a disruption in the RTK signaling survival pathway that is commonly associated with GBM. DYR726 performed favorably against Avapritinib (clinical trials for gliomas are beginning at Stanford) and when treated against FDA-approved combination treatment Dabrafenib+Trametinib (for 2% of glioma patients), it performed identically, suggesting that DYR726 can inhibit the cells at the same rate as the new FDA drug. Within patient-derived cell lines, it behaves identically to Buparlisib, and DYR726 has been shown to outperform AstraZenecas’ Osimertinib (which was significantly less potent) and Tafinlars’ Dabrafenib (displayed no effect). Based on the significance of efficacy, this series is on the CRUK Glasgow Cancer Center grant renewal as a potential Phase 0 within the next five years. Citation Format: Alessandra Fistrovich, Vasudah Tandon, Carly Cabel, Laura Basantes, Curtis Thorne, William Montfort, Sourav Banerjee, Christopher Hulme. The development of pleotropic small molecule kinase inhibitors (SKMIs) for the treatment of glioblastoma multiforme (GBM) [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 3100.

Abstract 5019: Next-generation DYRK1A inhibitors as a new therapeutic approach for the treatment of hematological malignancies

Abstract Background: The dual-specificity tyrosine-regulated kinase 1A (DYRK1A) can phosphorylate multiple targets involved in key cellular processes that have been associated with the hallmarks of cancer, including proliferation, survival, cell cycle regulation and the DNA damage response1. In addition, compounds that inhibit DYRK1A have demonstrated anti-tumor activity both in vitro and in vivo. However, until recently, there was a paucity of potent, specific, and bioavailable DYRK1A inhibitors that did not target the related cdc2-like kinases (CLKs) and glycogen synthase kinase 3β. In this study, we describe a set of next-generation DYRK1A inhibitors and used them to evaluate the specific contribution of DYRK1A activity to tumor biology and evaluate the therapeutic potential of DYRK1A inhibition in cancer. Methods: DYRK1A expression was analyzed in The Cancer Genome Atlas and Cancer Cell Line Encyclopedia databases. Cancer cell growth dependencies were evaluated by DepMap analysis. Cell-based target engagement (TE) was assessed by NanoBRETTM. Cell viability was assayed with CellTiter-Glo® following 4-day treatment with DYRK1A inhibitors. Synergy was calculated using Chalice software. Pharmacokinetics (PK) were evaluated in rodents and Tumor Growth Inhibition (TGI) in nude mice. Preliminary toxicology was assessed in a 14 day rat study. Results: DYRK1A expression and a systematic DYRK dependencies analysis in human cancers revealed that hematological malignancies, Small Cell Lung and Ovarian cancers rely on DYRK1A expression for their proliferation. A series of potent (biochemical IC50s 3nM to 10nM, TE 19 nM to 134 nM) and highly selective ATP-competitive DYRK1A inhibitors were designed with minimal inhibition of the rest of the kinome (1%-2.3% of kinases inhibited > 90%) Cancer cell lines (n=160) were treated with DYRK1A inhibitors and those belonging to hematological lineages were among the most sensitive (P<0.01). In vitro combination of DYRK1A inhibitors with the anti-leukemia agent venetoclax revealed high synergy in 3 out of the 5 AML cell lines tested (synergy score >20). Compounds that showed good oral bioavailability and plasma exposures in rodents were selected for further characterization in vivo. In the MV-4-11 xenograft model, mice treated daily with 25mg/kg, or 50mg/kg of 2 different DYRK1A showed 72-99% TGI compared to vehicle. Preclinical toxicity profile in rats showed next generation DYRK1A inhibitors were well tolerated. Concluding Remarks: these studies support further evaluation of DYRK1A inhibitors as a therapeutic strategy for some cancers, especially hematological malignancies including AML. 1 Boni, J.; Rubio-Perez, C.; López-Bigas, N.; Fillat, C.; de la Luna, S. The DYRK Family of Kinases in Cancer: Molecular Functions and Therapeutic Opportunities. Cancers 2020, 12, 2106. https://doi.org/10.3390/cancers12082106 Citation Format: Matthew C. Jarvis, Gopi Mittapalli, Emily Creger, Chelsea Nora, Maureen Ibanez, Deepti Bhat, Brian Hofilena, Josh Stewart, Elizabeth A. McMillan, Hadi Falahatpisheh, Chi-Ching Mak, Michael White, John S. Hill, Carine Bossard. Next-generation DYRK1A inhibitors as a new therapeutic approach for the treatment of hematological malignancies. [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 5019.

Comparative Efficacy and Selectivity of Pharmacological Inhibitors of DYRK and CLK Protein Kinases.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) play a large variety of cellular functions and are involved in several diseases (cognitive disorders, diabetes, cancers, etc.). There is, thus, growing interest in pharmacological inhibitors as chemical probes and potential drug candidates. This study presents an unbiased evaluation of the kinase inhibitory activity of a library of 56 reported DYRK/CLK inhibitors on the basis of comparative, side-by-side, catalytic activity assays on a panel of 12 recombinant human kinases, enzyme kinetics (residence time and Kd), in-cell inhibition of Thr-212-Tau phosphorylation, and cytotoxicity. The 26 most active inhibitors were modeled in the crystal structure of DYRK1A. The results show a rather large diversity of potencies and selectivities among the reported inhibitors and emphasize the difficulties to avoid "off-targets" in this area of the kinome. The use of a panel of DYRKs/CLKs inhibitors is suggested to analyze the functions of these kinases in cellular processes.

Rational design and appraisal of selective Cdc2-Like kinase 1 (Clk1) inhibitors as novel autophagy inducers for the treatment of acute liver injury (ALI)

Autophagy inducers are promising agents for treating certain medical illnesses, while no safe autophagy inducers are in clinical applications. Cdc2-like kinase 1 (Clk1) inhibitors induce autophagy efficiently; however, most Clk1 inhibitors lack selectivity, especially against Dyrk1A kinase. Herein, we report a series of 1H-pyrrolo[2,3-b]pyridin-5-amine derivatives as novel Clk1 inhibitors. Through detailed structural modification and structure-activity relationship studies, compound 10ad shows potent and selective inhibition for Clk1, with an IC50 value of 5 nM and over 300-fold selectivity for Dyrk1A. Related kinase screening also validates the selectivity of compound 10ad. Furthermore, compound 10ad potently induces autophagy in vitro and exhibits significant hepatoprotective effects in the acute liver injury model induced by acetaminophen (paracetamol). In general, due to the excellent potency and selectivity, compound 10ad was worth further investigation in the treatment of autophagy-related diseases.

SGC-CLK-1: A chemical probe for the Cdc2-like kinases CLK1, CLK2, and CLK4

Small molecule modulators are important tools to study both basic biology and the complex signaling of protein kinases. The cdc2-like kinases (CLK) are a family of four kinases that have garnered recent interest for their involvement in a diverse set of diseases such as neurodegeneration, autoimmunity, and many cancers. Targeted medicinal chemistry around a CLK inhibitor hit identified through screening of a kinase inhibitor set against a large panel of kinases allowed us to identify a potent and selective inhibitor of CLK1, 2, and 4. Here, we present the synthesis, selectivity, and preliminary biological characterization of this compound – SGC-CLK-1 (CAF-170). We further show CLK2 has the highest binding affinity, and high CLK2 expression correlates with a lower IC50 in a screen of multiple cancer cell lines. Finally, we show that SGC-CLK-1 not only reduces serine arginine-rich (SR) protein phosphorylation but also alters SR protein and CLK2 subcellular localization in a reversible way. Therefore, we anticipate that this compound will be a valuable tool for increasing our understanding of CLKs and their targets, SR proteins, at the level of phosphorylation and subcellular localization.