Casein Kinase Research Articles

Inhibitors of CK2 Kinase for the Treatment of Gliomas and Other Brain Tumors

Protein kinase CK2 has become a target of experimental antiglioma therapies as laboratory data are almost uniformly favorable and the number of synthetized CK2 inhibitors is rapidly growing. The evidence of their use for other brain tumors is on the increase as well. Great expectations are entrusted in naturally occurring compounds capable of inhibiting CK2 kinase. These compounds are extracted and purified by means of biochemistry methods and are amenable for innovative drug delivery systems as well. CK2 kinase inhibitors have been proven suitable for combined therapies with other investigational antiglioma agents and treatment modalities. However a greater share of efforts should be undertaken towards inhibiting functions relatively specific for glial tumors including infiltrative growth and invasiveness or maintenance of glioma initiating cells. Many of these function appear to converge on mTOR and JAK/STAT pathways which are being meticulously studied in this respect. Protein kinase CK2 holds therapeutic promise especially when combined with other agents aimed at molecular signatures of gliomas.

Structural Investigations on 2-Amidobenzimidazole Derivatives as New Inhibitors of Protein Kinase CK1 Delta

Protein kinase CK1δ (CK1δ) is a serine-threonine/kinase that modulates different physiological processes, including the cell cycle, DNA repair, and apoptosis. CK1δ overexpression, and the consequent hyperphosphorylation of specific proteins, can lead to sleep disorders, cancer, and neurodegenerative diseases. CK1δ inhibitors showed anticancer properties as well as neuroprotective effects in cellular and animal models of Parkinson’s and Alzheimer’s diseases and amyotrophic lateral sclerosis. To obtain new ATP-competitive CK1δ inhibitors, three sets of benzimidazole-2-amino derivatives were synthesized (1–32), bearing different substituents on the fused benzo ring (R) and diverse pyrazole-containing acyl moieties on the 2-amino group. The best-performing derivatives were those featuring the (1H-pyrazol-3-yl)-acetyl moiety on the benzimidazol-2-amino scaffold (13–32), which showed CK1δ inhibitor activity in the low micromolar range. Among the R substituents, 5-cyano was the most advantageous, leading to a compound endowed with nanomolar potency (23, IC50 = 98.6 nM). Molecular docking and dynamics studies were performed to point out the inhibitor–kinase interactions.

Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4

Piwi-interacting RNAs (piRNAs) are genomically encoded small RNAs that engage Piwi Argonaute proteins to direct mRNA surveillance and transposon silencing. Despite advances in understanding piRNA pathways and functions, how the production of piRNA is regulated remains elusive. Here, using a genetic screen, we identify casein kinase II (CK2) as a factor required for piRNA pathway function. We show that CK2 is required for the localization of PRG-1 and for the proper localization of several factors that comprise the ‘upstream sequence transcription complex’ (USTC), which is required for piRNA transcription. Loss of CK2 impairs piRNA levels suggesting that CK2 promotes USTC function. We identify the USTC component twenty-one-U fouled-up 4 (TOFU-4) as a direct substrate for CK2. Our findings suggest that phosphorylation of TOFU-4 by CK2 promotes the assembly of USTC and piRNA transcription. Notably, during the aging process, CK2 activity declines, resulting in the disassembly of USTC, decreased piRNA production, and defects in piRNA-mediated gene silencing, including transposons silencing. These findings highlight the significance of posttranslational modification in regulating piRNA biogenesis and its implications for the aging process. Overall, our study provides compelling evidence for the involvement of a posttranslational modification mechanism in the regulation of piRNA biogenesis.

Regulation of cancer progression by CK2: an emerging therapeutic target.

Casein kinase II (CK2) is an enzyme with pleiotropic kinase activity that catalyzes the phosphorylation of lots of substrates, including STAT3, p53, JAK2, PTEN, RELA, and AKT, leading to the regulation of diabetes, cardiovascular diseases, angiogenesis, and tumor progression. CK2 is observed to have high expression in multiple types of cancer, which is associated with poor prognosis. CK2 holds significant importance in the intricate network of pathways involved in promoting cell proliferation, invasion, migration, apoptosis, and tumor growth by multiple pathways such as JAK2/STAT3, PI3K/AKT, ATF4/p21, and HSP90/Cdc37. In addition to the regulation of cancer progression, increasing evidence suggests that CK2 could regulate tumor immune responses by affecting immune cell activity in the tumor microenvironment resulting in the promotion of tumor immune escape. Therefore, inhibition of CK2 is initially proposedas apivotal candidate for cancer treatment. In this review, we discussed the role of CK2 in cancer progression and tumor therapy.

ERK-activated CK-2 triggers blastema formation during appendage regeneration.

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal-regulated kinase (ERK)-activated casein kinase 2 (CK-2), which has not been previously implicated in appendage regeneration, triggers blastema formation during leg regeneration in the American cockroach, Periplaneta americana. After amputation, CK-2 undergoes rapid activation through ERK-induced phosphorylation within blastema cells. RNAi knockdown of CK-2 severely impairs blastema formation by repressing cell proliferation through down-regulating mitosis-related genes. Evolutionarily, the regenerative role of CK-2 is conserved in zebrafish caudal fin regeneration via promoting blastema cell proliferation. Together, we find and demonstrate that the ERK-activated CK-2 triggers blastema formation in both cockroach and zebrafish, helping explore initiation factors during appendage regeneration.

Abstract 5117: CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia

Abstract Survival for pre-B cell Acute lymphoblastic leukemia (B ALL) has improved greatly, however. High risk (HR) subgroups continue to result in significant mortality and morbidity of pediatric oncology patients. Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a lethal disease, often associated with high-risk clinical features. T-ALL therapy has made less improvement than B ALL and continues to represent a significant number of high-risk outcomes. Novel treatment strategies are required to overcome chemotherapy resistance and improve morbidity for HR B ALL. The development of treatments that can overcome chemotherapy resistance is essential for improving survival in T-ALL. Deletions of IKZF1, resulting in decreased IKAROS function, are present in High-risk ALL patients. Casein kinase 2 (CK2), a protein kinase upregulated in ALL, phosphorylates IKAROS preventing DNA binding reducing transcriptional regulation in ALL patients. Past studies have shown that pharmacologic inhibition of CK2 with CX-4945 rescues IKAROS function as a transcriptional regulator. Receptor tyrosine kinase c-KIT is transcriptionally regulated by IKAROS and is often upregulated in cancer. The purpose of this study was to determine whether restoration of IKAROS via CX-4945 would act in a synergistic manner with c-KIT inhibitor imatinib in ALL.Methods/results B-ALL Nalm6 cells were treated with imatinib over 72-hours to establish the half-maximal inhibitory concentration (IC50). The IC50 was used to determine the concentration range of imatinib to be used in combination with CX-4945 in a 72-hour synergy analysis of cytotoxicity.. Cell viability was measured using WST-1 cell proliferation assay. The data were analyzed using Calcusyn and synergy was defined as a combination index below 0.85. The study was repeated to determine a more precise range of synergy. The cytotoxicity of single drug c-KIT inhibitor, Imatinib, in Nalm6 B-ALL yielded an IC50 of 25uM while CK2 inhibitor CX-4945 resulted in 4uM. When a combination was administered with a constant dose of CX-4945 at 4uM and varying concentrations of imatinib, synergy was observed at multiple dose combinations. Conclusion: The data shows that CK2 inhibitor, CX-4945, and c-KIT inhibitor, imatinib, exhibit a synergistic therapeutic effect. Imatinib is indicated for use in Philadelphia positive (Ph+) acute lymphoblastic leukemia so future studies will include testing CX-4945/imatinib synergy in Ph+ BALL where we expect to see more potent synergistic effect. Citation Format: Daniel Gary Bogush, Joseph Schramm, Katarina Dovat, Yali Ding, Chingakham Singh, Sinisa Dovat. CK2 inhibiton exhibits synergistic effect in acute lymphoblastic leukemia [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 5117.

Abstract 3163: (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic colorectal cancer

Abstract Proteomic networks are crucial for cellular homeostasis maintenance and, indeed, their aberrant regulation is implicated in cancer development and progression. Understanding alterations underlying these pathways, provides a way to design therapeutic interventions in human cancer. Improvements in mass spectrometry analysis have unlocked our potential to unveil cancer vulnerabilities. Here, we performed (phospho)proteomic analysis and ex-vivo drug testing on a cohort of refractory metastatic colorectal cancer (mCRC) tissues and matched patients-Derived Organoids (PDO) to identify new putative druggable biomarkers. The comparison between the proteome and phosphoproteome of 10 mCRCs tissues with their matched PDOs normalized to a pool of 6 colon mucosa tissues highlighted a strong overall correlation (R= 0.57 and R=0.6, respectively) suggesting a good level of similarity. Applying GSEA on our proteomic dataset, we identified up-regulation in MYC, cell cycle and MTORC1 signaling pathways in mCRC tissues and PDO, while kinase enrichment analysis highlighted an increase in Casein Kinase II (CKII) activity, an important cell cycle regulator. Our ex-vivo experiments showed that silmitasertib-mediated CKII inhibition reduced the cell viability in our PDO models, but with an overall modest effect. Analysis revealed that, even though not statistically significant, high Wnt signaling score correlated with an higher resistance to silmitasertib treatment (R=0.61; p=0.08). Previous works highlighted that MAPK activity is increased after silmitasertib treatment and responsible for resistance mechanisms to CKII inhibition. Strikingly, the combined trametinib-mediated MEK blockade and CKII blockade resulted in a synergistic interaction leading to enhanced antitumor activity, especially in PDO harboring high Wnt (R=0.74; p=0.022) and cell cycle (R=0.69; p=0.04) pathway activity. Moreover, the combined treatment strongly reduced S6 activity and RAPTOR expression, overcoming the significant effect exerted by the single agents. Surprisingly, none of them altered baseline MAPK activity, suggesting that silmitasertib and trametinib synergize their activity via inactivating the MTORC1-S6 axis in an ERK-independent manner. We are currently integrating genomic and transcriptomic data to better characterize the observed drug synergy. Taken together, our findings propose the silmitasertib and trametinib combination as a new therapeutic opportunity for refractory mCRC patients and strengthen the use of PDO as a suitable model for drug screening. Citation Format: Mattia Marinucci, Gina Faye Boot, Lara Zaidi, Cinzia Esposito, Mairene Coto Llerena, Savas Soysal, Otto Kolmar, Charlotte K Y Ng, Salvatore Piscuoglio. (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic colorectal cancer [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 3163.

Abstract 3271: The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia

Abstract Casein Kinase II (CK2), a growth-promoting non-oncogene, is frequently over-expressed in pediatric high-risk B-cell acute lymphoblastic leukemia (B-ALL) leading to proliferation and survival. CK2 phosphorylates the tumor suppressor IKZF1 (IKAROS) resulting in a loss-of DNA-binding activity. Loss of IKZF1 function is associated with poor prognosis in high-risk B-ALL. As such, inhibition of CK2 using silmitasertib (CX-4945), to restore IKZF1 function, is being therapeutically investigated in high-risk B-ALL. CK2 also phosphorylates/activates the mammalian ceramide synthases to increase production of the pro-apoptotic sphingolipid, ceramide (Cer). Therefore, we hypothesized that inhibition of CK2 with CX-4945 would exert both pro-death (restoration of IKAROS function) and pro-survival (decreased Cer production) effects on B-ALL cells. Moreover, this suggests that targeting the sphingolipid metabolic pathway in tandem with CX-4945 could be a novel strategy for treatment of high-risk B-ALL. To this end, we examined the effects of CX-4945 on sphingolipid levels and the enzymes of the sphingolipid metabolic pathway. Unexpectedly, we determined that CX-4945 dose-dependently increased expression of ceramide synthase 1 (CERS1) and acid ceramidase (AC) proteins, in an IKZF1 dependent manner. Increased CERS1 expression/production of C18:0 Cer has been linked to enhanced mitophagy (i.e., lysosomal clearance of damaged mitochondria) in head and neck squamous cell carcinoma. Induction of mitophagy by CX-4945 was confirmed by western blot analysis of mitofusin-1 and Drp-1 levels. Up-regulation of AC, a lysosomal protein, metabolizes C18:0 Cer to sphingosine for recycling and prevents Cer-induced lysosomal membrane permeabilization. Thus, induction of mitophagy and metabolic breakdown of Cer could potentially reduce the apoptotic efficacy of CX-4945 in B-ALL cell lines. We, therefore, tested whether inhibition of AC would synergize with CX-4945 to enhance B-ALL cell death. Indeed, synergy was observed with the AC inhibitor, SACLAC, in multiple cell lines. Consistent with the observation of synergy between CX-4945 and SACLAC, the combination also enhanced cleavage of PARP indicating an enhancement of cell death. We are currently testing the efficacy of a combination of CX-4945 and AC inhibitors in cell-line derived xenograft models of human B-ALL. Together, these findings indicate that simultaneous targeting of CK2 and AC represents a rationally selected combinatorial therapeutic strategy that overcomes the ability of cancer cells to adapt to single agent targeted therapies by altering pro- and/or anti-apoptotic pathways. Citation Format: Jeremy A. Hengst, Diwakar Bastihalli Tukaramrao, Todd Fox, Arati Sharma, Dhimant Desai, Sinisa Dovat. The casein kinase 2/IKZF1 axis attenuates ceramide accumulation through up-regulation of acid ceramidase: Implications for use of CK2 inhibitors in high-risk pediatric B-cell acute lymphoid leukemia [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 3271.

Abstract 6254: Casein kinase II (CK2) expression in pediatric solid tumors

Abstract Despite recent advances, high-risk neuroblastoma (NB) and sarcomas account for 15% and 5% of all pediatric cancer deaths, respectively. Many patients attain remission, but approximately half of patients relapse. Genomic alterations such as MYCN amplification and ALK mutations have been shown to be important in prognosis and treatment. Despite improvements, approximately 20% of patients have refractory or recurrent disease after therapy, underscoring the need for further genomic understanding and new approaches to treatment.CK2 is a serine/threonine kinase that regulates numerous cellular processes such as cell growth, proliferation, and survival. Overexpression of CK2 is associated with a poor prognosis in several adult solid tumors, making it a potential target of interest in pediatric solid tumors. This study aims to investigate CK2 expression among patients with NB, Ewing sarcoma (EWS), and rhabdomyosarcoma (RMS) and its correlation with patient survival. MethodsPatients were enrolled in NMTRC008/009: “Feasibility Trial Using Molecular Guided Therapy for Patients with Relapsed/Refractory Childhood Cancer” and BCC-001. WES and RNA-Seq were performed at the Translational Genomics Research Institute and Sema4 or Caris respectively. RNA expression levels were compared to a normal whole-body reference and a pediatric cancer reference. Differential expression data were interpreted in the context of systems biology.ResultsTumor WES was performed on 297 patients (212 NB, 50 EWS, 35 RMS) and RNA-Seq on 284 patients (210 NB, 39 EWS, 35 RMS). The most common mutations or copy number alterations were seen in MYCN, ATRX, ALK, CDKN2A, and chr17q+ in NB; EWSR1-FLI1 fusion in EWS; PAX3-FOXO1 fusion in RMS. RNA expression analysis was performed to understand MYCN-driver status by covariance and interactions and to identify subsets of patients with high/low expression of CK2 in different tumors. CK2 bimodal expression is seen across various cancers and normal tissues, implying distinct sub-groups exist in the data. In NB, overexpression of the CK2 gene with statistical significance was found in MYCN-amplified and chr17q+ tumors. A positive and negative correlation of the CK2 gene is seen with several target genes like MYCN, TRIM71, LIN28B, and HMGA2, JAK2, AR, STAT3, EGFR, LEF1, and NFKB2, respectively. In EWS and RMS, subgroups are identified with high and low expression of the CK2 gene.ConclusionGenomic sequencing of pediatric solid tumors showed a low mutation burden. RNA analysis resulted in the identification of potential novel therapeutic targets involving CK2 pathways. High expression of CK2 correlates with MYCN status. Further analysis of patient molecular subgroups is ongoing. Citation Format: Abhinav Beeravally Nagulapally, Chandrika Behura, Divya Gandra, Giselle Saulnier Sholler. Casein kinase II (CK2) expression in pediatric solid tumors [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 6254.

Abstract 251: MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis

Abstract To establish and maintain functional and properly formed tissues, cells must integrate cues from their environment into fate decisions. External cues—from other cells and the extracellular matrix for instance—must be met with appropriate responses to ensure cell fate decisions that control lineage plasticity and favor proper development, maintenance, or regeneration of tissues while suppressing neoplastic transformation. MCAM (CD146; MUC18; S-endo-1; and Gicerin) is an integral membrane glycoprotein whose misexpression is associated with a variety of epithelial cancers, including breast cancer. However, the mechanisms by which MCAM conveys extracellular cues into malignant cellular outputs is not well understood. Our observation that MCAM is normally expressed in transplantable fetal and adult mammary stem cells (MaSCs) with multilineage potential, suggested it could control mammary cell state plasticity in both neoplastic and normal mammary tissue. Using a plastic, mouse mammary tumor cell line derived from the MMTV-PyMT mouse (Py230) that has been reported to exhibit mammary stem cell and luminal progenitor features, we tested the role of Mcam in controlling the tumorigenicity and lineage/cell-state relationships of mammary tumor cells. We found that Mcam knockdown in Py230 cells increases STAT3 expression, activation, and downstream target transcription, leading to a shift in the predominant cell state away from a luminal progenitor state toward more differentiated alveolar and basal phenotypes. Mechanistically, these changes involve altered activity of casein kinase II (CK2) against multiple substrates including STAT3 and PTEN. This effect may be mediated by direct coupling and sequestration of CK2 to various substrates via the cytoplasmic tail of MCAM. We have also found that inhibition of Ck2 or Stat3 reversed the transcriptional state change elicited by Mcam knockdown. In grafted tumors in vivo, this state switch correlates with reduced overall tumorigenicity and attenuated expression of Sox10, a gatekeeper of neural crest-like invasive features. In the normal gland, we hypothesize that this activity regulates multilineage stem cell potential upon transplantation and multilineage regenerative potential across lactation/involution cycles as we found reciprocal expression patterns for MCAM and STAT3 in alveolar mammary epithelial cells. These data point to an integral role for MCAM in lineage plasticity of normal mammary epithelial cells that is co-opted during transformation to potentiate tumor cell plasticity, progression, and evasion of targeted cancer therapies. Citation Format: Brooke L. Gates, Ozlen Balcioglu, David W. Freeman, Berhane M. Hagos, Benjamin T. Spike. MCAM controls mammary cell state in luminal progenitor-derived breast cancer and alveologenesis [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 251.

Abstract 3338: Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer

Abstract A novel, potent and highly selective orally bioavailable small molecule inhibitor of Casein Kinase 2α (CK2α) has been developed and validated in pre-clinical models of wnt-driven colorectal carcinoma; and is poised to enter clinical development.CK2α is considered as a positive regulator of wnt signaling and tumorigenesis. The wnt pathway is known to be sensitive to and amplified by CK2α activity at multiple nodes and can be inhibited by loss of CK2α function. The human genetics of colorectal cancer (CRC) have been well characterized and approximately 80% tumors are identified as being wnt pathway mutation driven. CRC tumors can be further stratified into consensus molecular subtypes (CMS), with iCMS2 representing 37% of CRC patients, being characterized by ongoing wnt pathway upregulation. Development of CK2α inhibitors as a targeted therapy for CRC is therefore mechanistically well aligned to the wnt signaling drive and may serve as a promising therapeutic strategy for this cancer. A novel small molecule binding site on CK2α has been exploited to design and develop an inhibitor with Ki of 95 pM and exquisite selectivity over the kinome. This inhibitor has been comprehensively characterized using in vitro, ex vivo and in vivo pharmacology studies. Cellular mechanism of action studies in CRC cell lines have quantified the potency of target engagement, inhibition of known CK2α phospho-sites, inhibition of tumorigenic wnt pathway activity, and induction of apoptosis. This CK2α inhibitor was also profiled in a high content screen of CRC patient-derived organoids established from various driver mutation backgrounds. Use of these ex vivo models has validated the potent cytotoxic activity of CK2α inhibition in tumor samples with wnt-driven characteristics and highlights the potential therapeutic efficacy of CK2α inhibition in this setting. In vivo CRC xenograft studies have confirmed anti-tumor efficacy and developed the understanding of pharmacokinetic pharmacodynamic relationship between free plasma concentration of drug and inhibition of CK2α-specific biomarker in the tumor. The preclinical data presented establishes the strong rationale for clinical development of a novel CK2α inhibitor for the treatment of locally advanced or metastatic solid tumors, with an emphasis on wnt-driven CRC. Citation Format: Darren Cawkill. Development of a CK2α inhibitor for treatment of wnt-driven colorectal cancer [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 3338.

Abstract 604: Targeting casein kinase 1 alpha (CK1 alpha) and transcriptional CDKs (CDK7/9) in human liposarcomas

Abstract Introduction: Liposarcomas (LPS) are rare mesenchymal cell malignancies of adipocytic origin that are diagnosed in more than 3500 patients in the US each year. The two most common subtypes, well-differentiated LPS (WDLPS) and dedifferentiated LPS (DDLPS), are characterized by extrachromosomal DNA amplifications harboring the MDM2 (100%) and CDK4 (90+%) genes, generally with wild type TP53. Management of metastatic or surgically unresectable LPS remains purely palliative. Recent clinical trials targeting MDM2 or CDK4/6 with small-molecule inhibitors have shown promise but have been hampered by dose-limiting toxicities. The development of new therapeutics is greatly needed to improve outcomes for patients with LPS. Results: To identify unique liposarcoma-specific vulnerabilities, we screened multiple human LPS cell lines for transcriptional CDK expression and found high levels of CDK7 and CDK9. We demonstrate that CDK9 inhibitors suppress LPS cell growth and induce apoptosis by decreasing MDM2 levels while inducing expression of p53. To enhance p53 activation in these cells, we screened for expression of known regulators of p53, including CK1α, whose inhibition has previously been shown to activate p53. We demonstrate that LPS cells express CK1α and that the cytotoxic effects of CDK9 inhibitors are enhanced upon CK1α depletion. These data led us to examine combined targeting of CK1α and CDK9 in LPS with the novel agent BTX-A51, which has previously been shown to inhibit CK1α, CDK9, as well as CDK7 with nanomolar efficacy in AML models. BTX-A51 potently reduces expression of MDM2 with marked induction of p53, resulting in profound apoptosis of LPS cells. Through CRISPR/Cas9-mediated p53 knockout, we establish that BTX-A51-mediated apoptosis is primarily p53-dependent. However, BTX-A51 also reduces expression of MCL1 and primes LPS cell lines and primary LPS cells for BIM-induced apoptosis, as demonstrated by BH3 profiling. Importantly, preliminary in vivo data in an LPS patient-derived xenograft model reveal that BTX-A51 is well-tolerated with tumor growth inhibition. Conclusions: Our results suggest that BTX-A51 has potent preclinical efficacy in treating LPS, primarily through inhibition of CK1α and CDK9. Future mechanistic studies will further clarify mechanisms of BTX-A51-mediated apoptosis, as well as the contribution of CDK7 inhibition to anti-tumor activity. Our data justify a planned clinical trial that will evaluate the efficacy of BTX-A51 in patients with advanced WDLPS or DDLPS. Citation Format: Renyan Liu, Nicole L. Solimini, Patrick Bhola, Timothy B. Branigan, Jie Hao, Xin Wang, Roshen Alharthi, Michael Yorsz, Shaili Soni, Cing-siang Hu, Irit Snir-Alkalay, Prafulla Gokhale, Anthony Letai, Yinon Yinon Ben-Neriah, George D. Demetri, Geoffrey I. Shapiro. Targeting casein kinase 1 alpha (CK1 alpha) and transcriptional CDKs (CDK7/9) in human liposarcomas [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 604.

CK2 activity is crucial for proper glucagon expression.

Protein kinase CK2 acts as a negative regulator of insulin expression in pancreatic beta cells. This action is mainly mediated by phosphorylation of the transcription factor pancreatic and duodenal homeobox protein 1 (PDX1). In pancreatic alpha cells, PDX1 acts in a reciprocal fashion on glucagon (GCG) expression. Therefore, we hypothesised that CK2 might positively regulate GCG expression in pancreatic alpha cells. We suppressed CK2 kinase activity in αTC1 cells by two pharmacological inhibitors and by the CRISPR/Cas9 technique. Subsequently, we analysed GCG expression and secretion by real-time quantitative RT-PCR, western blot, luciferase assay, ELISA and DNA pull-down assays. We additionally studied paracrine effects on GCG secretion in pseudoislets, isolated murine islets and human islets. In vivo, we examined the effect of CK2 inhibition on blood glucose levels by systemic and alpha cell-specific CK2 inhibition. We found that CK2 downregulation reduces GCG secretion in the murine alpha cell line αTC1 (e.g. from 1094±124 ng/l to 459±110 ng/l) by the use of the CK2-inhibitor SGC-CK2-1. This was due to a marked decrease in Gcg gene expression through alteration of the binding of paired box protein 6 (PAX6) and transcription factor MafB to the Gcg promoter. The analysis of the underlying mechanisms revealed that both transcription factors are displaced by PDX1. Ex vivo experiments in isolated murine islets and pseudoislets further demonstrated that CK2-mediated reduction in GCG secretion was only slightly affected by the higher insulin secretion after CK2 inhibition. The kidney capsule transplantation model showed the significance of CK2 for GCG expression and secretion in vivo. Finally, CK2 downregulation also reduced the GCG secretion in islets isolated from humans. These novel findings not only indicate an important function of protein kinase CK2 for proper GCG expression but also demonstrate that CK2 may be a promising target for the development of novel glucose-lowering drugs.

Preclinical Targeting of the PGRMC1-CK2 Axis with Silmitasertib: A Potential Strategy for Lung Adenocarcinoma Therapy.

Progesterone receptor membrane component 1 (PGRMC1) is a pleiotropic protein over-expressed in lung adenocarcinoma (LUAD). The precise molecular mechanisms underlying the signature motif of Casein kinase (CK2) presence in PGRMC1 and their role in LUAD remain unclear. X-ray crystallographic structure for CK2 and PGRMC1 from the PubChem database was obtained and subjected to protein-protein interaction (PPI) analysis to identify their interactions. In addition, the CK2 inhibitor - Silmitasertib was also utilised to understand the interaction between PGRMC1-CK2. The PPI complex (PGRMC1-CK2) and the PPI-ligand interaction analysis and their Molecular Dynamics (MD) studies revealed the stability of their interactions and critical amino acid contacts within the 5Ǻ vicinity of the CK2 signature motif "T/S-x-x-E/D". Moreover, in-vitro colony formation assay, migration assay, and gene expression analysis using quantitative Real-time PCR revealed that Silmitasertib (IC50-2.5 μM) was highly influential in suppressing the PGRMC1-CK2 expression axis. In conclusion, our study infers that PGRMC1-CK-2 axis inhibition could be a potential therapeutic option to limit the promotion and progression of lung cancer.

A major gene for chilling tolerance variation in Indica rice codes for a kinase OsCTK1 that phosphorylates multiple substrates under cold.

Rice is susceptible to chilling stress. Identifying chilling tolerance genes and their mechanisms are key to improve rice performance. Here, we performed a genome-wide association study to identify regulatory genes for chilling tolerance in rice. One major gene for chilling tolerance variation in Indica rice was identified as a casein kinase gene OsCTK1. Its function and natural variation are investigated at the physiological and molecular level by its mutants and transgenic plants. Potential substrates of OsCTK1 were identified by phosphoproteomic analysis, protein-protein interaction assay, in vitro kinase assay, and mutant characterization. OsCTK1 positively regulates rice chilling tolerance. Three of its putative substrates, acidic ribosomal protein OsP3B, cyclic nucleotide-gated ion channel OsCNGC9, and dual-specific mitogen-activated protein kinase phosphatase OsMKP1, are each involved in chilling tolerance. In addition, a natural OsCTK1 chilling-tolerant (CT) variant exhibited a higher kinase activity and conferred greater chilling tolerance compared with a chilling-sensitive (CS) variant. The CT variant is more prevalent in CT accessions and is distributed more frequently in higher latitude compared with the CS variant. This study thus enables a better understanding of chilling tolerance mechanisms and provides gene variants for genetic improvement of chilling tolerance in rice.

Casein kinase 2 phosphorylates and induces the SALL2 tumor suppressor degradation in colon cancer cells

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family’s most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Modulating the Activity of the Human Organic Cation Transporter 2 Emerges as a Potential Strategy to Mitigate Unwanted Toxicities Associated with Cisplatin Chemotherapy.

Cisplatin (CDDP) stands out as an effective chemotherapeutic agent; however, its application is linked to the development of significant adverse effects, notably nephro- and ototoxicity. The human organic cation transporter 2 (hOCT2), found in abundance in the basolateral membrane domain of renal proximal tubules and the Corti organ, plays a crucial role in the initiation of nephro- and ototoxicity associated with CDDP by facilitating its uptake in kidney and ear cells. Given its limited presence in cancer cells, hOCT2 emerges as a potential druggable target for mitigating unwanted toxicities associated with CDDP. Potential strategies for mitigating CDDP toxicities include competing with the uptake of CDDP by hOCT2 or inhibiting hOCT2 activity through rapid regulation mediated by specific signaling pathways. This study investigated the interaction between the already approved cationic drugs disopyramide, imipramine, and orphenadrine with hOCT2 that is stably expressed in human embryonic kidney cells. Regarding disopyramide, its influence on CDDP cellular transport by hOCT2 was further characterized through inductively coupled plasma isotope dilution mass spectrometry. Additionally, its potential protective effects against cellular toxicity induced by CDDP were assessed using a cytotoxicity test. Given that hOCT2 is typically expressed in the basolateral membrane of polarized cells, with specific regulatory mechanisms, this work studied the regulation of hOCT2 that is stably expressed in Madin-Darby Canine Kidney (MDCK) cells. These cells were cultured in a matrix to induce the formation of cysts, exposing hOCT2 in the basolateral plasma membrane domain, which was freely accessible to experimental solutions. The study specifically tested the regulation of ASP+ uptake by hOCT2 in MDCK cysts through the inhibition of casein kinase II (CKII), calmodulin, or p56lck tyrosine kinase. Furthermore, the impact of this manipulation on the cellular toxicity induced by CDDP was examined using a cytotoxicity test. All three drugs-disopyramide, imipramine, and orphenadrine-demonstrated inhibition of ASP+ uptake, with IC50 values in the micromolar (µM) range. Notably, disopyramide produced a significant reduction in the CDDP cellular toxicity and platinum cellular accumulation when co-incubated with CDDP. The activity of hOCT2 in MDCK cysts experienced a significant down-regulation under inhibition of CKII, calmodulin, or p56lck tyrosine kinase. Interestingly, only the inhibition of p56lck tyrosine kinase demonstrated the capability to protect the cells against CDDP toxicity. In conclusion, certain interventions targeting hOCT2 have demonstrated the ability to reduce CDDP cytotoxicity, at least in vitro. Further investigations in in vivo systems are warranted to ascertain their potential applicability as co-treatments for mitigating undesired toxicities associated with CDDP in patients.

Abstract 1002 Regulation of Cisplatin Resistance in Lung Cancer Cells by Casein Kinase 2, CD44, and IGFBP-3

Abstract 1002 Regulation of Cisplatin Resistance in Lung Cancer Cells by Casein Kinase 2, CD44, and IGFBP-3

A novel PD-1/PD-L1 pathway-related seven-gene signature for the development and validation of the prognosis prediction model for breast cancer.

Breast cancer (BC/BRCA) is the most common carcinoma in women. The average 5-year survival rate of BC patients with stage IV disease is 26%. A considerable proportion of patients still do not receive effective therapy. It is an unmet need to identify novel biomarkers for BC patients. Herein, we evaluated whether the programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) status is associated with the clinical outcomes of BC, based on data from The Cancer Genome Atlas (TCGA). Clinical and transcriptome data of BC patients were obtained from TCGA dataset, and prognostic genes in BC patients were identified, as well as the PD-1/PD-L1 pathway mainly associating with the BC patients. Following the execution of the consensus clustering algorithm, BC patients were segregated into two clusters, and subsequent investigation of the potential mechanisms between them was carried out. A comparison of ferroptosis and N6-methyladenosine (m6A) was conducted between the two groups with the greatest difference in prognosis. Based on least absolute shrinkage and selection operator (LASSO) analysis, a signature associated with the PD-1/PD-L1 pathway was developed, and the prognosis outcome and the predictive accuracy of the signature model were further assessed. Prognostic genes in BC patients were studied using TCGA data and it was found that the PD-1/PD-L1 pathway was most associated with the BC patients. Then, a low-risk (C1) group and a high-risk (C2) group of BC patients were constructed based on a PD-1/PD-L1 pathway-related signature. The functional analyses suggested that the underlying mechanisms between these groups were mainly associated with immune-related pathways. We found that ferroptosis and m6A were significantly different between the two groups. A PD-1/PD-L1 pathway-related gene signature was further developed to predict survival of BC patients, including 7 genes [mitogen-activated protein kinase kinase 6 (MAP2K6), NF-kappa-B inhibitor alpha (NFKBIA), NFKB Inhibitor Epsilon (NFKBIE), Interferon gamma (IFNG), Toll/interleukin-1 receptor domain-containing adapter protein (TIRAP), IkappaB kinase (CHUK), and Casein kinase 2 alpha 3 gene (CSNK2A3)]. The receiver operating characteristic (ROC) curves were analyzed to further assess the prognostic values of these 7 genes. The 1-, 3-, and 5-year values of the areas under the curve (AUCs) for overall survival were 0.651, 0.658, and 0.653 in this seven gene signature model, respectively. PD-1/PD-L1 pathway-related subtypes of BC were identified, which were closely associated with the immune microenvironment, the ferroptosis status, and m6A in BC patients. The gene signature involved in the PD-1/PD-L1 pathway might help to make a distinction and predict prognosis in BC patients.

Abstract 1639 Chloroplast protein phosphatase SLP1 counters protein kinase, casein kinase 2

Abstract 1639 Chloroplast protein phosphatase SLP1 counters protein kinase, casein kinase 2