Cdc2-like Kinase Research Articles

Discovery of CLKs inhibitors for the treatment of non-small cell lung cancer

Targeted inhibition of the Wnt pathway is a promising strategy for treating NSCLC. CDC2-like kinase 2 (CLK2), a dual-specificity kinase responsible for phosphorylating serine/arginine-rich (SR) proteins, can modulate Wnt signaling through the alternative splicing of Wnt target genes, making CLK2 an attractive therapeutic target for NSCLC. In this study, we report the synthesis, optimization, and evaluation of CLK2 inhibitors that effectively suppress the proliferation of NSCLC cells, with the identification of the lead compound LBM22. Notably, compound LBM22 demonstrated potent inhibition of CLK2 (IC50 = 3.9 nM), leading to broad suppression of NSCLC cells proliferation and induction of apoptosis. Furthermore, LBM22 dose-dependently suppressed SR protein phosphorylation (pSRSF4, pSRSF5, and pSRSF6) in NSCLC cells, while downregulating the expression of Wnt pathway-related proteins (p-β-catenin, Axin 2, and c-Myc) as well as anti-apoptotic proteins (Bcl-2 and Mcl-1). Additionally, significant antiproliferative activity was observed for LBM22 in 3D cultured H1975OR cells. In conclusion, LBM22 emerges as a promising CLK2 inhibitor for the treatment of NSCLC.

8213 A Splicing Modulator Inhibits Proliferation and Steroid Production of H295R Adrenocortical Cancer Cells

Abstract Disclosure: T. Tanaka: None. K. Ohe: None. T. Yanase: None. S. Kodama: None. Adrenocortical carcinoma (ACC) is a rare cancer, and treatment options are limited. The anti-tumor drug CX-4945 (Silmitasertib) is an inhibitor of Casein kinase 2 (CK2). It also inhibits cdc2-like kinase 2 (CLK2). CLK2 phosphorylates Serine/arginine rich splicing factors (SRSFs)/SR proteins and enhances exonic splicing enhancer (ESE) recognition during the pre-RNA splicing process. Anti-tumor agents targeting RNA splicing have been developed recently, and CLK inhibitors have shown anti-tumor effects against blood cancers. In this study, we analyzed the impact of CX-4945 on NCI-H295R cells. CX-4945 inhibited cell proliferation of H295R (IC50 = 6.4 µM, 72hr), and cortisol and aldosterone secretion in vitro. siRNA-induced CK2 inhibition did not alter H295R proliferation and steroid secretion. CX-4945 (150 mg/kg/day oral, 28 days) significantly reduced tumor weight in mice inoculated with H295R subcutaneously on the back. High-density microarray analysis showed reduced Nuclear receptor 5A1 (NR5A1) expression levels, indicating a possible splicing variant. RT-PCR, using a primer set that amplified the coding region, detected short variants of NR5A that were multiple exon-skipped and were not induced by suppression of CK2. The results of western blotting using an antibody that recognizes phospho-epitope of SRSFs/SR proteins showed that the phosphorylation of SRSF4 was reduced in H295R treated with CX-4945. These results suggest that CX-4945 inhibits CLK2 and decreases phosphorylated SRSF4 and ESE recognition, resulting in NR5A1 multiple exon-skipping. The prognosis of ACC patients with high NR5A1 expression levels is poor, independent of stage. CX-4945 may induce NR5A1 multiple exon-skipping, reduce full-length functional NR5A1, and suppress ACC growth and steroidogenesis, as a possible adjuvant in tackling this devastating cancer. Presentation: 6/1/2024

8213 A Splicing Modulator Inhibits Proliferation And Steroid Production Of H295R Adrenocortical Cancer Cells

Abstract Disclosure: T. Tanaka: None. K. Ohe: None. T. Yanase: None. S. Kodama: None. Adrenocortical carcinoma (ACC) is a rare cancer, and treatment options are limited. The anti-tumor drug CX-4945 (Silmitasertib) is an inhibitor of Casein kinase 2 (CK2). It also inhibits cdc2-like kinase 2 (CLK2). CLK2 phosphorylates Serine/arginine rich splicing factors (SRSFs)/SR proteins and enhances exonic splicing enhancer (ESE) recognition during the pre-RNA splicing process. Anti-tumor agents targeting RNA splicing have been developed recently, and CLK inhibitors have shown anti-tumor effects against blood cancers. In this study, we analyzed the impact of CX-4945 on NCI-H295R cells. CX-4945 inhibited cell proliferation of H295R (IC50 = 6.4 µM, 72hr), and cortisol and aldosterone secretion in vitro. siRNA-induced CK2 inhibition did not alter H295R proliferation and steroid secretion. CX-4945 (150 mg/kg/day oral, 28 days) significantly reduced tumor weight in mice inoculated with H295R subcutaneously on the back. High-density microarray analysis showed reduced Nuclear receptor 5A1 (NR5A1) expression levels, indicating a possible splicing variant. RT-PCR, using a primer set that amplified the coding region, detected short variants of NR5A that were multiple exon-skipped and were not induced by suppression of CK2. The results of western blotting using an antibody that recognizes phospho-epitope of SRSFs/SR proteins showed that the phosphorylation of SRSF4 was reduced in H295R treated with CX-4945. These results suggest that CX-4945 inhibits CLK2 and decreases phosphorylated SRSF4 and ESE recognition, resulting in NR5A1 multiple exon-skipping. The prognosis of ACC patients with high NR5A1 expression levels is poor, independent of stage. CX-4945 may induce NR5A1 multiple exon-skipping, reduce full-length functional NR5A1, and suppress ACC growth and steroidogenesis, as a possible adjuvant in tackling this devastating cancer. Presentation: 6/1/2024

Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes. We discovered CDC-like kinase 1 (CLK1) is aberrantly spliced to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation. Inhibition of CLK1 with Cirtuvivint significantly decreased both cell viability and proliferation in the pediatric HGG KNS-42 cell line. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability with concurrent differential expression of 78 cancer genes and differential splicing at functional sites in 193 cancer genes. Our findings highlight a dependency of pediatric HGGs on CLK1 and represent a promising therapeutic strategy.

Disruption of cotranscriptional splicing suggests RBM39 is a therapeutic target in acute lymphoblastic leukemia

AbstractThere are only a few options for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL), thus, this is a major area of unmet medical need. In this study, we reveal that the inclusion of a poison exon in RBM39, which could be induced by both CDK9 or CDK9 independent cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, CDC-like kinases (CMGC) kinase inhibition, is recognized by the nonsense-mediated messenger RNA decay pathway for degradation. Targeting this poison exon in RBM39 with CMGC inhibitors led to protein downregulation and the inhibition of ALL growth, particularly in relapsed/refractory B-ALL. Mechanistically, disruption of cotranscriptional splicing by the inhibition of CMGC kinases, including DYRK1A, or inhibition of CDK9, which phosphorylate the C-terminal domain of RNA polymerase II (Pol II), led to alteration in the SF3B1 and Pol II association. Disruption of SF3B1 and the transcriptional elongation complex altered Pol II pausing, which promoted the inclusion of a poison exon in RBM39. Moreover, RBM39 ablation suppressed the growth of human B-ALL, and targeting RBM39 with sulfonamides, which degrade RBM39 protein, showed strong antitumor activity in preclinical models. Our data reveal that relapsed/refractory B-ALL is susceptible to pharmacologic and genetic inhibition of RBM39 and provide 2 potential strategies to target this axis.

Targeting the CLK2/SRSF9 splicing axis in prostate cancer leads to decreased ARV7 expression.

In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant-7 (ARV7) is clinically the most relevant and has a distinct 3' untranslated region (3'UTR) compared to the AR full-length variant, suggesting a unique post-transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3'UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine-rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2-like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti-proliferative effects in enzalutamide-treated or -naive PC models. Thus, targeting aberrant alternative splicing at the 3'UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI-resistant PC.

A phase 1 single-arm, open-label study of emavusertib (CA-4948) in combination with azacitidine and venetoclax in patients (pts) with acute myeloid leukemia (AML) in complete response (CR) with measurable residual disease (MRD).

TPS6587 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 3 onco-proteins by emavusertib may address the unmet need for novel therapies in AML. Clinical studies with emavusertib monotherapy have demonstrated a significant reduction in AML blasts with clinical and molecular responses in pts with relapsed or refractory AML (Metzeler 2022). Azacitidine + venetoclax (aza+ven) is an approved therapy for newly diagnosed pts with AML, unfit for intensive chemotherapy. Pre-clinical studies have demonstrated that emavusertib in combination with aza+ven has synergistic and antileukemic effects. In the VIALE-A study, composite CR (CRc) (CR, CRh, or CRi) in the absence of MRD of <1 residual blast/1000 leukocytes (MRD negative [MRD−]) resulted in longer duration of response (DOR), event-free survival, and overall survival (OS) compared with AML pts who achieved CRc but were MRD+ (Pratz, 2022). Therefore, adding emavusertib to aza+ven in CR or CRh, MRD+ pts may convert MRD status without significant toxicity and become a potential new regimen in front-line therapy. Methods: This is a single-arm, open-label Phase 1 study of emavusertib in combination with first line aza+ven in AML pts ≥60 years of age who achieved CR or CRh with MRD+ status based on local testing. The primary objective is to determine a safe and tolerable dosing schedule for the triple combination. Secondary objectives include determining the MRD conversion rate, pharmacokinetics, DOR, and OS. The study will enroll approximately 24 pts at 5 to 10 sites globally. Pts will have received aza+ven 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 aza+ven regimen. Key exclusion criteria include residual toxicities and significant comorbidities. In the first cohort, the starting emavusertib dose is 200 mg BID for 7 days per cycle of 28 days. The duration of emavusertib treatment will extend to 14 and 21 days, in subsequent cohorts. The pts will continue triple treatment (emavusertib+aza+ven) until consent withdrawal, disease progression, intolerable toxicity, or not achieving MRD- after 6 cycles of treatment. Enrollment to cohort 1 began in December 2023. (EU CT Number 2023-505828-58). Clinical trial information: 2023-505828-58.

Pharmacodynamics of venetoclax plus cirtuvivint a first-in-class splice variant regulator.

e15093 Background: NSC835563 (SM08502, cirtuvivint) is a first-in-class pan CDC-like kinase (CLK) and dual specificity tyrosine kinase (DYRK) inhibitor that targets alternative splicing of genes in multiple pathways and modifies their protein expression. Preclinical screening of hematologic malignancy models has shown sensitivity to cirtuvivint, leading to proposed clinical trials in AML in combinations with venetoclax. We performed pharmacodynamic studies to identify probable mechanisms of combination activity in AML models. Methods: In vivo tolerability, efficacy and pharmacodynamic (PD) studies were performed in two AML xenograft models, MV411 and KG1a in NSG mice, at 6.25-25 mg/kg PO cirtuvivint and 25 mg/kg PO venetoclax. Body weights (BW) and tumor volumes were recorded periodically to assess toxicity and anti-tumor efficacy. Tumor tissue samples were collected at 6 and 24 h post venetoclax to assess PD response. Multiplexed analysis of apoptosis pathway proteins/protein-protein complexes was performed by clinically validated assays (PMID 26446940). Results: Cirtuvivint alone at high doses (25 mg/kg) caused tumor regressions in MV411 but only a modest tumor growth inhibition (TGI) in KG1a, a resistant AM model. Importantly, the lowest dose of 6.25 mg/kg cirtuvivint combined with 25 mg/kg venetoclax led to tumor regression in the MV411 model and a moderate TGI in KG1a. Cirtuvivint alone caused a transient 25% reduction (p<0.05) in Mcl-1 levels at 6 h post dose in MV411. The venetoclax combination triggered a sustained (for 24 h) 50-75% disruption of Bcl-2 and Mcl-1 complexes with Bax and Bim, resulting in >20-fold increased mitochondrial Bax/Bak heterodimer and >10-fold increased cytosolic cleaved caspase-3. In KG1a, combination treatment resulted in only modest (<20%) changes in biomarkers. Overall, biomarkers provided evidence of the combination MoA. Conclusions: Venetoclax and cirtuvivint in combination effectively induced tumor regressions at low tolerable doses in an AML model. Mitochondrial Bax/Bak heterodimer provided a sensitive biomarker of combination activity. Cirtuvivint contributed its combinatorial effect primarily through disruption of Bim complexes and inhibition of Mcl-1. PD biomarkers identified in this study can confirm MoA of drug combination in ongoing clinical trials.

In silico identification of a novel Cdc2-like kinase 2 (CLK2) inhibitor in triple negative breast cancer.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

AR-V7 expression facilitates accelerated G2/M phase transition in castration-resistant prostate cancer

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

Abstract CT150: 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

Abstract 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. Citation Format: Reinhard von Roemeling, Adolfo De La Fuente, Claudio Cerchione, Sebastian Scholl, Jan Moritz Middeke, Gaurav S. Choudhary, Maria Lamar, Steven Angelides, Uwe Platzbecker. 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 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT150.

Abstract CT110: A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in solid tumor cohorts

Abstract Background: Aberrant splicing contributes to the progression of cancer but lacks targeted drugs. CTX-712 is a potent oral CDC2-like kinase (CLK) inhibitor affecting RNA splicing, CTX-712 demonstrated potent inhibition of proliferation in human tumor cell lines and elicited robust anti-tumor activity in xenograft models. This study aimed to evaluate CTX-712’s maximum tolerated dose (MTD), dose-limiting toxicity (DLT), safety, pharmacokinetics (PK)/pharmacodynamics (PD) profiles, and preliminary efficacy in patients with advanced solid tumors. Methods: This study consisted of a dose escalation cohort (A) and three dose expansion cohorts (C, D and E) for solid tumors. The dose escalation cohort (A) was initiated with accelerated titration and then switched to a 3+3 design (10, 20, 40, 70, 105, 140 and 175 mg twice a week [TW]). The dosing regimens for dose expansion cohorts were 105 mg TW (C), 70 mg TW (D), and 105 mg once a week (QW) (E). Results: As of November 20, 2023, a total of 46 patients with solid tumors were enrolled in the dose escalation cohort (16 patients) and dose expansion cohorts (30 patients total). In the dose escalation cohort, CTX-712 was administered at a dose of 10/20/40/70 mg TW (n=1 each), 105/175 mg TW (n=3 each) and 140 mg TW (n=6). DLTs were observed in 2 patients (140 mg TW [platelet count decreased, hypokalemia], 175 mg TW [dehydration]) and MTD was determined to be 140 mg TW. Among the safety analysis population (n=46), median treatment duration was 77 days (Range 1-413+ days); 1 patient is ongoing, and 45 patients discontinued. The common any-grade related adverse events (AEs) (≥30%) were nausea (93.5%), vomiting (65.2%), diarrhea (54.3%), decreased appetite (45.7%) and blood creatinine increase (32.6%). The most common related Grade 3 or higher AE was platelet count decrease (6.5%). Among all the patients (n=46), partial responses were observed in 4 (8.7%) patients (40 (n=1)/70 (n=2)/105 (n=1) mg TW cohorts), who were all patients with ovarian cancer; the median treatment duration of ovarian cancer (n=14) was 84 days (Range 1-413+ days). 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 (S6K and THAP9-AS1) demonstrated mRNA splicing modification induced by CTX-712. Conclusions: CTX-712 demonstrated an acceptable safety profile in patients with solid tumors. Additionally, CTX-712 demonstrated favorable clinical efficacy for patients with ovarian cancer and further investigation is warranted. Clinical trial information: jRCT2080224127 Citation Format: Noboru Yamamoto, Takafumi Koyama, Yuki Katsuya, Jun Sato, Toshio Shimizu, Yasushi Tanoue, Maki Yamamoto, Hirokazu Tozaki, Eiji Takahara, Shingo Shoji, Akio Mizutani, Daisuke Morishita, Robert W. Oda, Hiroshi Miyake, Kan Yonemori. A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in solid tumor cohorts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT110.

Abstract CT115: A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in a hematologic malignancies cohort

Abstract Background: Mutations in genes encoding RNA splicing factors such as SF3B1, SRSF2, U2AF1, and ZRSR2, are common genetic aberrations in many forms of myeloid neoplasms. CTX-712 is a first-in-class, orally available, highly potent, and selective small molecule inhibitor of CDC2-like kinase (CLK), a key regulator of RNA splicing. Preclinical data show that CTX-712 demonstrated anti-proliferative activity in in-vitro and in-vivo models derived from hematologic malignancies. A phase I study of CTX-712 was conducted to evaluate the safety and the preliminary efficacy of CTX-712 in patients with relapsed or refractory acute myeloid leukemia (AML) and higher-risk myelodysplastic syndrome (MDS). Methods: The objectives of this cohort were to evaluate the dose-limiting toxicity (DLT), safety, pharmacokinetic (PK)/pharmacodynamic (PD) profiles, and preliminary efficacy of CTX-712 in patients with hematologic malignancies. A 3+3 design was used in two dose levels (70 mg and 105 mg twice a week (TW)) based on the safety data from the previous dose escalation cohort in patients with solid tumors. Results: As of data cutoff on November 20, 2023, a total of 14 patients (12 AML and 2 MDS) were enrolled in 70 mg TW (n=8) and 105 mg TW dose levels (n=6). DLT of Grade 4 pneumonia was observed in 1 of 3 DLT-evaluable patients in the 105 mg TW group. In the 70 mg TW group, no DLT was observed in 6 DLT-evaluable patients. Among the safety analysis population (n=14), median treatment duration was 76 days (Range 15-974 days). At data cutoff, treatment was ongoing in 1 patient and discontinued in 13 patients. The common any-grade adverse events (AEs) (≥30%) were diarrhea (57.1%) and nausea (42.9%). The most common Grade 3 or higher AE were white blood cell count decreased and tumor lysis syndrome (14.3%). In AML patients (n=12), complete remission (CR) was observed in 3 patients (25.0%), and CR with incomplete hematologic recovery (CRi) was observed in 1 patient (8.3%). The median time to remission was 67 days (Range 29-113 days), and the median duration of response was 281 days (Range 56-924 days). In MDS patients (n=2), CR was observed in 1 patient (50.0%). PK/PD profiles of the hematologic malignancies dose escalation cohort were comparable with those of the solid tumor cohort at the same dosage. Conclusions: CTX-712 demonstrated a manageable and tolerable safety profile and showed anti-tumor efficacy in patients with hematologic malignancies. Currently, a Phase I/II Study of CTX-712 in relapsed/refractory AML and higher risk MDS is ongoing using a new tablet formulation in the United States. Clinical trial information: jRCT2080224127 Citation Format: Hisayuki Yokoyama, Noriko Fukuhara, Koji Ando, Hiroatsu Iida, Takahiro Yamauchi, Suguru Fukuhara, Koji Izutsu, Yasushi Tanoue, Maki Yamamoto, Hirokazu Tozaki, Eiji Takahara, Shingo Shoji, Akio Mizutani, Daisuke Morishita, Robert W. Oda, Hiroshi Miyake, Noboru Yamamoto. A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in a hematologic malignancies cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT115.

CDC-like kinase 3 deficiency aggravates hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

Hypoxia-induced cardiomyocyte apoptosis is one major pathological change of acute myocardial infarction (AMI), but the underlying mechanism remains unexplored. CDC-like kinase 3 (CLK3) plays crucial roles in cell proliferation, migration and invasion, and nucleotide metabolism, however, the role of CLK3 in AMI, especially hypoxia-induced apoptosis, is largely unknown. The expression of CLK3 was elevated in mouse myocardial infarction (MI) models and neonatal rat ventricular myocytes (NRVMs) under hypoxia. Furthermore, CLK3 knockdown significantly promoted apoptosis and inhibited NRVM survival, while CLK3 overexpression promoted NRVM survival and inhibited apoptosis under hypoxic conditions. Mechanistically, CLK3 regulated the phosphorylation status of AKT, a key player in the regulation of apoptosis. Furthermore, overexpression of AKT rescued hypoxia-induced apoptosis in NRVMs caused by CLK3 deficiency. Taken together, CLK3 deficiency promotes hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

Targeting the Cdc2-like kinase 2 for overcoming platinum resistance in ovarian cancer.

Platinum resistance represents a major barrier to the survival of patients with ovarian cancer (OC). Cdc2-like kinase 2 (CLK2) is a major protein kinase associated with oncogenic phenotype and development in some solid tumors. However, the exact role and underlying mechanism of CLK2 in the progression of OC is currently unknown. Using microarray gene expression profiling and immunostaining on OC tissues, we found that CLK2 was upregulated in OC tissues and was associated with a short platinum-free interval in patients. Functional assays showed that CLK2 protected OC cells from platinum-induced apoptosis and allowed tumor xenografts to be more resistant to platinum. Mechanistically, CLK2 phosphorylated breast cancer gene 1 (BRCA1) at serine 1423 (Ser1423) to enhance DNA damage repair, resulting in platinum resistance in OC cells. Meanwhile, in OC cells treated with platinum, p38 stabilized CLK2 protein through phosphorylating at threonine 343 of CLK2. Consequently, the combination of CLK2 and poly ADP-ribose polymerase inhibitors achieved synergistic lethal effect to overcome platinum resistance in patient-derived xenografts, especially those with wild-type BRCA1. These findings provide evidence for a potential strategy to overcome platinum resistance in OC patients by targeting CLK2.

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4608.

Abstract 5944: Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers

Abstract Aberrant alternative splicing is a newly recognized hallmark of cancer, that has been shown to play a critical role in tumorigenesis, cancer progression, and therapeutic resistance via multiple mechanisms, including increased proliferation, decreased apoptosis, promoted migration, enhanced metastatic potential, and induced evasion of immune surveillance. Serine and arginine-rich splicing factors (SRSFs) are RNA-binding proteins (RBPs) that regulate constitutive and alternative splicing. SRSFs are often mutated or overexpressed in cancers, resulting in widespread alterations in splicing patterns. The Cdc2-like kinase (CLK) family and dual-specificity tyrosine-regulated kinase (DYRK) phosphorylate SRSFs, influencing the assembly of spliceosome machinery, exon recognition, and splicing. Therefore, targeting CLK/DYRK kinases can modulate cancer specific splicing isoforms, opening avenues for new therapeutic interventions. BH-30236 was designed as a novel orally bioavailable, ATP-competitive, macrocyclic inhibitor of CLK with IC50s of 0.134, 0.165, and 0.446 nM against CLK1, CLK2, and CLK4, respectively in enzymatic kinase assays. At clinically relevant concentrations, BH-30236 also inhibited DYRK1A/1B/2, proviral insertion site of moloney murine leukemia virus kinase 3 (PIM3), and FMS-like tyrosine kinase 3 (FLT3) with IC50s of 0.111, 0.148, 0.562, 0.115 and 0.248 nM, respectively. In cancer cells, BH-30236 impaired the phosphorylation of SRSFs, Tau and 4EBP1, the direct downstream substrates of CLK, DYRK, and PIM kinases with IC50s of 40-60 nM, ~50 nM, and ~80 nM, respectively. Furthermore, BH-30236 potently inhibited the FLT3 phosphorylation with an IC50 of 0.16 nM. Overall, BH-30236 regulated alternative splicing by primarily inducing skipped exons in favor of anti-tumor isoforms, leading to cancer cell death and growth suppression in a broad panel of cancer cell lines and in vivo efficacy studies. For example, BH-30236 potently inhibited cell proliferation with an IC50 of 0.62 nM in FLT3-ITD positive MV-4-11 cells and achieved complete tumor regression in the MV-4-11 cell-derived xenograft tumor model, even after dose cessation for 4 weeks. In MV-4-11 cells, BH-30236 increased pro-apoptosis splicing variant BCLxS, downregulated RNA expression of BCL2, MCL1, and AML stem cell markers CD33 and CD123. In addition, BH-30236 has demonstrated good human ADME and preclinical safety profiles. Collectively, the preclinical study results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors, as a single agent or in combination with other therapies. Citation Format: J. Jean Cui, Ping Jiang, Wei Deng, Dayong Zhai, Nancy Ling, Danan Li, Zhenping Wang, Yue Hu, Levan Darjania, Jeff Whitten, Evan Rogers, Eugene Rui. Discovery of BH-30236: A novel macrocyclic CLK inhibitor targeting alternative splicing in cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5944.

Abstract 617: BH-30236, a novel macrocyclic CLK inhibitor modulating RNA splicing, demonstrates potent inhibition of cancer cell growth in a broad panel of cancer cell lines

Abstract Alternative splicing (AS) is a primary mechanism for mRNA transcript diversification and protein expression regulation. In cancer, altered mRNA splicing promotes oncogenic transformation, induces metastasis, and confers resistance to cancer treatment. Mutations or imbalanced expression/activity of splicing factors (SF), such as serine-arginine-rich SFs (SRSFs), often result in deregulation of RNA splicing and tumor progression. CDC2-like kinases (CLK) and dual-specificity tyrosine-regulated kinase (DYRK) are key regulators of AS via phosphorylation of SRSFs. BH-30236 is a novel macrocyclic ATP-competitive inhibitor of CLK1/2/4. It also inhibits DYRK1/2, provirus integration site for moloney leukemia virus 3 (PIM3) and FMS-like tyrosine kinase 3 (FLT3) at clinically relevant concentrations. The inhibitory activity of BH-30236 against CLK, DYRK, PIM3 and FLT3 in cancer cells was investigated via the phosphorylation inhibition of SRSFs, Tau, 4EBP1 and FLT3, respectively. A panel of 119 hematological and solid tumor cell lines were used to investigate the spectrum of BH-30236 against cancer cell growth. To understand the mechanism of anti-cancer cell growth, the effect of BH-30236 on AS and protein expression of key tumor related biomarkers were examined. In conclusion, BH-30236 effectively inhibited pSRSF (IC50 40-60 nM in IMR-32 cells), pTau (IC50 ~50 nM in SH-SY5Y cells), p4EBP1 (IC50 ~80 nM in MM1S cells) and pFLT3-ITD (IC50 0.16 nM in MV-4-11 cells). BH-30236 demonstrated broad inhibition of cancer cell growth with a median IC50 of 85.2 nM (range 0.55-393 nM) across 12 tumor types. BH-30236 showed great efficacy against cell lines derived from hematological malignancy (median IC50 23.34 nM), neuroblastoma (median IC50 25.73 nM), breast cancer (median IC50 83.80 nM), colon cancer (median IC50 85.17 nM), and lung cancer (median IC50 102.65 nM). Furthermore, BH-30236 demonstrated synergy with KRAS inhibitors in KRAS mutant cell lines, with EGFR inhibitors in RBM10 mutant H1975 cells, and with BCL2 inhibitor Venetoclax in MOLM-13 cells. Mechanistically, BH-30236 modulated AS by increasing pro-apoptotic and anti-proliferative splicing variants of key factors, such as BCL2L1, S6K, and BCLAF1. Meanwhile, BH-30236 downregulated RNA expression of SRSFs and modulated BCL2 family to increase apoptosis. These results strongly support the clinical applications of the novel multikinase CLK inhibitor BH-30236 in hematological malignancies and solid tumors as a single agent or in combination with other therapies. Citation Format: Ping Jiang, Danan Li, Nancy Ling, Dayong Zhai, Wei Deng, Eugene Rui, J. Jean Cui. BH-30236, a novel macrocyclic CLK inhibitor modulating RNA splicing, demonstrates potent inhibition of cancer cell growth in a broad panel of cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 617.

Abstract 5105: Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas

Abstract Pediatric brain cancer is the leading cause of disease-related mortality in children. With a paucity of therapeutically targetable alterations, there is a pressing need to identify effective strategies for many of these tumors. In this study, we systematically examine aberrant alternative splicing in pediatric brain tumors from the Open Pediatric Brain Tumor Atlas, and additional tumors from the Children's Brain Tumor Network and the Pacific Pediatric Neuro-Oncology Consortium (n = 1831). Notably, high-grade gliomas (HGGs), including those with and without H3 histone mutations, were enriched in the upper quartile of all tumors with high splicing burden (SB). HGGs demonstrated the greatest SB heterogeneity of all tumor types and we identified a total of 19,229 aberrant splicing events in 8,577 genes in midline tumors compared to non-tumor brainstem controls (ΔPSI >= |.20|, p < 0.05). We next integrated protein annotations from the UniProt Knowledgebase to assess and prioritize differential alternative splicing events affecting protein function. This approach led to the discovery of 3,754 splice variants in 5,036 genes resulting in the gain or ablation of functional sites. The protein kinase CDC-like kinase 1 (CLK1), a recognized master splicing factor and cell-cycle modulator, exhibited aberrant splicing of exon 4, whereby inclusion promoted the gain of two known phosphorylation sites. Additionally, we observed a strong correlation between CLK1 splicing, its own RNA expression and proteomic activity of the Serine/Arginine-rich (SR) family of splicing factors. The distribution of exon 4 splicing was significantly different (p = 0.019) between tumors with low vs high splicing burden; tumors with high splicing burden had increased exon 4 inclusion. Further, we performed experimental modulation of CLK1 exon 4 aberrant splicing in the brain tumor cell line KNS42 and demonstrated that forced exon 4 skipping leads to reduced CLK1 protein abundance and significantly reduces cell proliferation (p < 0.01 vs. control). Following bulk RNA-Seq analysis of these cell lines, we identified that forced CLK1 exon 4 skipping resulted in 4,518 dysregulated genes enriched for known cancer pathways such as mitotic spindle, E2F targets, and G2M checkpoint. Our data suggest a role for CLK1 in driving transcriptional dysregulation in pediatric high-grade gliomas and uncover a potential therapeutic vulnerability. Citation Format: Ammar S. Naqvi, Priyanka Seghal, Ryan Corbett, Komal Rathi, Brian Ennis, Bo Zhang, Miguel Brown, Daniel Miller, Adam Kraya, Joseph Dybas, Katharina Hayer, Shehbeel Arif, Zhuangzhuang Geng, Antonia Chroni, Aditya Lahiri, Karina Conkrite, Madison Hollawell, Sharon Diskin, Peter Madsen, Jessica B. Foster, Mateusz P. Koptyra, Andrei Thomas-Tikhonenko, Adam Resnick, Phillip J. Storm, Jo Lynne Rokita. Aberrant CLK1 splicing is a key dependency factor in pediatric high-grade gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5105.

Structure-Guided Discovery of Potent and Selective CLK2 Inhibitors for the Treatment of Knee Osteoarthritis.

Osteoarthritis is the most common joint disorder. However, there are no disease-modifying drugs approved for OA treatment. CDC2-like kinase 2 (CLK2) could modulate Wnt signaling via alternative splicing of Wnt target genes and further affect bone differentiation, chondrocyte function, and inflammation, making CLK2 an attractive target for OA therapy. In this study, we designed and synthesized a series of highly potent CLK2 inhibitors based on Indazole 1. Among them, compound LQ23 showed more elevated inhibitory activity against CLK2 than the lead compound (IC50, 1.4 nM) with high CLK2/CLK3 selectivity (>70-fold). Furthermore, LQ23 showed outstanding antiosteoarthritis effects in vitro and in vivo, with the roles specific in decreased inflammatory cytokines, downregulated cartilage degradative enzymes, and increased joint cartilage via suppressing CLK2/Wnt signaling pathway. Overall, these data support LQ23 as a potential candidate for intra-articular knee OA therapy, leveraging its unique mechanism of action for targeted treatment.