Germinal Center Kinase Research Articles

Role of Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Signaling in Liver and Metabolic Diseases.

MAP4K4 is a serine/threonine protein kinase belonging to the germinal center kinase subgroup of sterile 20 protein family of kinases. MAP4K4 has been involved in regulating multiple biologic processes and a plethora of pathologies, including systemic inflammation, cardiovascular diseases, cancers, and metabolic and hepatic diseases. Recently, multiple reports have indicated the upregulation of MAP4K4 expression and signaling in hyperglycemia and liver diseases. This review provides an overview of our current knowledge of MAP4K4 structure and expression, as well as its regulation and signaling, specifically in metabolic and hepatic diseases. Reviewing these promising studies will enrich our understanding of MAP4K4 signaling pathways and, in the future, will help us design innovative therapeutic interventions against metabolic and liver diseases using MAP4K4 as a target. SIGNIFICANCE STATEMENT: Although most studies on the involvement of MAP4K4 in human pathologies are related to cancers, only recently its role in liver and other metabolic diseases is beginning to unravel. This mini review discusses recent advancements in MAP4K4 biology within the context of metabolic dysfunction and comprehensively characterizes MAP4K4 as a clinically relevant therapeutic target against liver and metabolic diseases.

Specific deletion of FHA domain containing SLMAP isoform in mice impacts embryonic development without affecting Hippo signaling. AP Dias, T Rehmani, M Salih BS Tuana

The tail-anchored membrane protein SLMAP3 has been proposed to negatively regulate Hippo signaling and impact cell proliferation. Proteomics have defined SLMAP in complex with STRIPAK a multiprotein interactome consisting of Germinal center kinases and PP2A phosphatase. Data also suggests that the N-terminal FHA domain of SLMAP3 can bind Hippo kinase (MST 1/2) while it‘s coiled-coil regions can integrate PP2A phosphatase to modulate the downstream phosphorylation of YAP/TAZ for cytoplasmic retention. Nuclear YAP/TAZ serve as coactivators of TEAD mediated transcription of genes involved in cell proliferation and organogenesis. The physiological importance of SLMAP3 and it‘s in vivo function in regulating Hippo and organ development remains to be determined. We report that mice with specific deletion of SLMAP3 isoform were embryonic lethal where most organs including muscle, brain, spinal cord, kidney, lungs, intestines, cartilage and bone exhibited severe developmental deficits. The SLMAP3 deficient embryos were ~30% smaller (p<0.05) with small organs compared to wild types. The analysis of Hippo components MST1/2 and YAP/TAZ indicated no changes in their phosphorylation status in embryos or tissue such as muscle and the neural tube due to the loss of SLMAP3. RNA-Seq of whole embryos showed no significant changes to downstream targets of Hippo signaling in SLMAP3-KO. In addition, SLMAP3 loss had no obvious effect on cell proliferation assessed with Ki-67/pH3 staining in embryogenesis. Mouse primary embryonic fibroblasts (MEFs) isolated from embryos deficient in SLMAP3 had no changes in YAP and MST1/2 phosphorylation or proliferation while RNA-seq analysis indicated no changes in TEAD regulated gene activity. Further, phospho-proteomics of MEF lysates revealed no changes in Hippo signaling in SLMAP3 loss, nor in pathways associated with cell proliferation or death. Similarly, knockout of SLMAP3 in myoblast displayed no changes in Hippo signaling or proliferation capacity. These data indicate that SLMAP3 is critically important for embryonic development through unique mechanisms and do not implicate the involvement of Hippo signaling. Supported by CIHR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 603: BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models

Abstract Introduction: Immuno-suppressive micro-environment and abnormal signaling pathways are crucial for the proliferation of tumor cells. Hematopoietic progenitor kinase 1 (HPK1) inhibitor plays important roles in activation of exhausted T cells and enhancement of anti-tumor immune responses. VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family, and is reported to be essential for Wnt signaling and colorectal cancer (CRC) proliferation and progression. Blockade of critical kinases in multiple signaling pathways inhibits the proliferation, invasion and metastasis of cancer cells. Therefore, intergradation of these functions in a small molecule serves as the next generation of immune-oncology small molecules for cancer therapy. Methods: BB5523 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. CDX and syngeneic tumor models in mice were conducted to evaluate the in vivo efficacy of BB5523 as a single agent. Results: BB5523 shows high inhibition potency on HPK1 with good selectivity against other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 kinase activity (IC50 = 3.3 nM) by BB5523 strongly suppresses the phosphorylation of the downstream biomarker protein SLP-76 (IC50 = 30 nM); and boosts the IL-2 secretion in purified human T cells and human peripheral blood mononuclear cells. BB5523 also shows good activities on VEGFR2 (IC50 = 1.6 nM) and TNIK (IC50 = 0.7 nM) which are crucial for the proliferation of cancer cells. In in vitro experiments, BB5523 significantly inhibits the proliferation of various tumor cell lines at sub-micromolar concentrations. PK profile of BB5523 shows good oral bioavailability (F > 80%). In preliminary safety evaluation, no abnormal symptoms were observed in mice dosed up to 100 mpk for 14 days. BB5523 showed significant tumor-growth inhibition efficacy in TE-8, JIMT-1, B16-F10 and MC38 tumor models. Conclusion: As a potent and oral available small molecule kinase inhibitor, BB5523 boosts anti-tumor immunity through HPK1 inhibition, while also blocking crucial signaling pathways in tumor micro-environment. BB5523 shows promising in vitro and in vivo efficacy with a well-tolerated safety profile. The preclinical study of BB5523 is in progress. Citation Format: Gongping Duan, Min Li, Puyong Zhang, Wei Hua, Enlun Hao, Shipeng Wang, Xiaoyi Ma, Junheng Wang, Lijie Wei, Xingmin Zhang. BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models [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 603.

Deficiency of germinal center kinase TRAF2 and NCK-interacting kinase (TNIK) in B cells does not affect atherosclerosis.

Atherosclerosis is the underlying cause of many cardiovascular diseases, such as myocardial infarction or stroke. B cells, and their production of pro- and anti-atherogenic antibodies, play an important role in atherosclerosis. In B cells, TRAF2 and NCK-interacting Kinase (TNIK), a germinal center kinase, was shown to bind to TNF-receptor associated factor 6 (TRAF6), and to be involved in JNK and NF-κB signaling in human B cells, a pathway associated with antibody production. We here investigate the role of TNIK-deficient B cells in atherosclerosis. ApoE-/-TNIKfl/fl (TNIKBWT) and ApoE-/-TNIKfl/flCD19-cre (TNIKBKO) mice received a high cholesterol diet for 10 weeks. Atherosclerotic plaque area did not differ between TNIKBKO and TNIKBWT mice, nor was there any difference in plaque necrotic core, macrophage, T cell, α-SMA and collagen content. B1 and B2 cell numbers did not change in TNIKBKO mice, and marginal zone, follicular or germinal center B cells were unaffected. Total IgM and IgG levels, as well as oxidation specific epitope (OSE) IgM and IgG levels, did not change in absence of B cell TNIK. In contrast, plasma IgA levels were decreased in TNIKBKO mice, whereas the number of IgA+ B cells in intestinal Peyer's patches increased. No effects could be detected on T cell or myeloid cell numbers or subsets. We here conclude that in hyperlipidemic ApoE-/- mice, B cell specific TNIK deficiency does not affect atherosclerosis.

Serine/threonine-protein kinase STK24 induces tumorigenesis by regulating the STAT3/VEGFA signaling pathway

Lung cancer is the most common cause of cancer-related death. Although anti-angiogenesis therapy has been effective in the treatment of nonsmall cell lung cancer (NSCLC), drug-resistance is a common challenge. Therefore, there is a need to develop new therapeutic strategies for NSCLC. Serine/threonine-protein kinase 24 (STK24), also known as MST3, belongs to the germinal center kinase III subfamily, and the biological function of STK24 in NSCLC tumorigenesis and tumor angiogenesis is still unclear. In this study, we demonstrated that STK24 was overexpressed in lung cancer tissues compared with normal lung tissues, and lung cancer patients with higher STK24 expression levels had shorter overall survival time. In addition, our invitro assays using A549 and H226cell lines revealed that the STK24 expression level of cancer cells was positively correlated with cancer cells proliferation, migration, invasion, and tumor angiogenesis ability; invivo assays also demonstrated that silencing of STK24 dramatically inhibited tumor progress and tumor angiogenesis. To investigate a mechanism, we revealed that STK24 positively regulated the signal transducer and activator of transcription 3 (STAT3)/vascular endothelial growth factor A (VEGFA) signaling pathway by inhibiting polyubiquitin-proteasomal-mediated degradation of STAT3. Furthermore, we performed invivo assays in BALB/c nude mice and invitro assays to show that STK24-regulated tumor angiogenesis depends on STAT3. These findings deepened our understanding of tumor angiogenesis, and the STK24/STAT3/VEGFA signaling pathway might be a novel therapeutic target for NSCLC treatment.

NDR kinase tricornered genetically interacts with Ccm3 and metabolic enzymes in Drosophila melanogaster tracheal development.

The Germinal Center Kinase III (GckIII) pathway is a Hippo-like kinase module defined by sequential activation of Ste20 kinases Thousand and One (Tao) and GckIII, followed by nuclear dbf2-related (NDR) kinase Tricornered (Trc). We previously uncovered a role for the GckIII pathway in Drosophila melanogaster tracheal (respiratory) tube morphology. The trachea form a network of branched epithelial tubes essential for oxygen transport, and are structurally analogous to branched tubular organs in vertebrates, such as the vascular system. In the absence of GckIII pathway function, aberrant dilations form in tracheal tubes characterized by mislocalized junctional and apical proteins, suggesting that the pathway is important in maintaining tube integrity in development. Here, we observed a genetic interaction between trc and Cerebral cavernous malformations 3 (Ccm3), the Drosophila ortholog of a human vascular disease gene, supporting our hypothesis that the GckIII pathway functions downstream of Ccm3 in trachea, and potentially in the vertebrate cerebral vasculature. However, how GckIII pathway signaling is regulated and the mechanisms that underpin its function in tracheal development are unknown. We undertook biochemical and genetic approaches to identify proteins that interact with Trc, the most downstream GckIII pathway kinase. We found that known GckIII and NDR scaffold proteins are likely to control GckIII pathway signaling in tracheal development, consistent with their conserved roles in Hippo-like modules. Furthermore, we show genetic interactions between trc and multiple enzymes in glycolysis and oxidative phosphorylation, suggesting a potential function of the GckIII pathway in integrating cellular energy requirements with maintenance of tube integrity.

MAP4K2 Inhibition Reinforces the Iberdomide Sensitivity in MM Cells By Inducing IKZF1 Degradation through a CRBN Independent Mechanism

MAP4K2 is predominantly and highly expressed in the germinal center of B cells, also called Germinal Center Kinase (GCK). Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. IKZF1 is a critical transcriptional factor regulating IRF4 and c-MYC transcription in MM, therefore we expect that IKZF1 degradation is one of the major mechanisms that contribute to the inhibition of MAP4K2 induced MM cell death. To address the role of IKZF1 degradation in MAP4K2 inhibition induced cytotoxicity in MM, we generated a mutant IKZF1 by substituting Glutamine Q146 to Histidine, which abrogates IKZF1 ubiquitination induced by CRBN. MM.1S cells were transduced with lentiviral constructs to overexpress flag-IKZF1WT or flag-IKZF1Q146H. Lenalidomide failed to induce a complete IKZF1-degradation in IKZF1Q146H MM.1S cells, indicating IMiDs induced IKZF1-degradation is mediated by CRBN binding and subsequent ubiquitination. In contrast, IKZF1 mutation did not rescue MM.1S from MAP4K2 inhibitor (TL4-12) induced IKZF1 downregulation. Proliferation assays further confirmed that IKZF1Q146H MM.1S cells were resistant to LEN induced cell growth inhibition, but not MAP4K2 inhibitor, demonstrating that MAP4K2 inhibition causes the degradation of IKZF1 and cell growth inhibition through a mechanism different from that of lenalidomide in myeloma cells. We further evaluated the effects of MAP4K2 silencing in IKZF1Q146H MM.1S cells. MAP4K2 knockdown resulted in significant inhibition of cell proliferation and increase of apoptotic cells in both cell lines (flag-IKZF1WT or flag-IKZF1Q146H MM.1S). Flag-IKZF1WT or flag-IKZF1Q146H were also overexpressed in IMiDs resistant (RPMI-8266) MM cell line by lentiviral infection, and no differences of MAP4K2 knockdown induced change of IKZF1 levels, cell proliferation, or apoptosis were observed between two cell lines. These data suggest that MAP4K2 inhibition induces anti-MM effects by targeting IKZF1 via an CRBN independent mechanism and subsequently overcomes IMiDs resistance. Our recent work also showed that the combination of iberdomide and MAP4K2 silencing led to synergetic anti-MM effects in vitro. In previous biochemical and structural studies, it was demonstrated that iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. To investigate the combined effects of Iberdomide with MAP4K2 silencing on MM tumor growth, we generated subcutaneous MM xenografts in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice using the inducible MAP4K2 shRNA MM.1S cells. Combination of Iberdomide with MAP4K2 silencing further enhanced tumor inhibition compared to the single treatment. Furthermore, we found that combination treatment significantly prolonged mice survival, compared to the single treatment. Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore will provide a novel strategy to manage the relapsed or refractory patients with multi-drug resistance.

MST4 negatively regulates type I interferons production via targeting MAVS-mediated pathway

BackgroundCytosolic RNA sensing can elicit immune responses against viral pathogens. However, antiviral responses must be tightly regulated to avoid the uncontrolled production of type I interferons (IFN) that might have deleterious effects on the host. Upon bacterial infection, the germinal center kinase MST4 can directly phosphorylate the adaptor TRAF6 to limit the inflammatory responses, thereby avoiding the damage caused by excessive immune activation. However, the molecular mechanism of how MST4 regulates virus-mediated type I IFN production remains unknown.MethodsThe expression levels of IFN-β, IFIT1, and IFIT2 mRNA were determined by RT-PCR. The expression levels of p-IRF3, IRF3, RIG-I, MAVS, and MST4 proteins were determined by Western blot. The effect of secreted level of IFN-β was measured by ELISA. The relationship between MST4 and MAVS was investigated by immunofluorescence staining and coimmunoprecipitation.ResultsIn this study, we reported that MST4 can act as a negative regulator of type I IFN production. Ectopic expression of MST4 suppressed the Poly (I:C) (polyino-sinic-polycytidylic acid)- and Sendai virus (SeV)-triggered production of type I IFN, while the knockdown of MST4 enhanced the production of type I IFN. Mechanistically, upon SeV infection, the MST4 competed with TRAF3 to bind to the 360–540 domain of MAVS, thereby inhibiting the TRAF3/MAVS association. Additionally, MST4 facilitated the interaction between the E3 ubiquitin ligase Smurf1 and MAVS. This promoted the K48-linked ubiquitination of MAVS, thereby accelerating the ubiquitin-mediated proteasome degradation of MAVS.ConclusionsOur findings showed that MST4 acted as a crucial negative regulator of RLR-mediated type I IFN production.-szD4SjHvt2BGoxb8QjZ8LVideo

GCKIII kinases in lipotoxicity: Roles in NAFLD and beyond.

Nonalcoholic fatty liver disease (NAFLD) is defined by excessive accumulation of lipid droplets within hepatocytes. The STE20‐type kinases comprising the germinal center kinase III (GCKIII) subfamily – MST3, MST4, and STK25 – decorate intrahepatocellular lipid droplets and have recently emerged as critical regulators of the initiation and progression of NAFLD. While significant advancement has been made toward deciphering the role of GCKIII kinases in hepatic fat accumulation (i.e., steatosis) as well as the aggravation of NAFLD into its severe form nonalcoholic steatohepatitis (NASH), much remains to be resolved. This review provides a brief overview of the recent studies in patient cohorts, cultured human cells, and mouse models, which have characterized the function of MST3, MST4, and STK25 in the regulation of hepatic lipid accretion, meta‐inflammation, and associated cell damage in the context of NAFLD/NASH. We also highlight the conflicting data and emphasize future research directions that are needed to advance our understanding of GCKIII kinases as potential targets in the therapy of NAFLD and its comorbidities. Conclusions: Several lines of evidence suggest that GCKIII proteins govern the susceptibility to hepatic lipotoxicity and that pharmacological inhibition of these kinases could mitigate NAFLD development and aggravation. Comprehensive characterization of the molecular mode‐of‐action of MST3, MST4, and STK25 in hepatocytes as well as extrahepatic tissues is important, especially in relation to their impact on carcinogenesis, to fully understand the efficacy as well as safety of GCKIII antagonism.

Quantification of adaptive forces on SNP rs1010211 due to viral zoonotic pathogens

Widespread genotyping of human populations in environmental homeostasis has created opportunities to quantify how environmental parameters affect the genomic distribution of variants in healthy populations. This represents an ongoing natural experiment upon the human species that can only be understood through developing models of adaptation. By examining the information dynamics of optimal SNP distributions within such populations, “adaptive forces” on genomic variants can be quantified through comparisons between different populations. To this end, we are performing double-blind scans of SNPs in order to ascertain any potential smooth functional relationships between the frequencies of the variants and changes in quantified environmental parameters. At present, we have sequentially examined more than twenty thousand SNPs (on chromosome 3) of nine homeostatic native populations for potential adaptive flagging of the variants as functions of 15 environmental parameters. Our first significant flag has related rs1010211 to viral pathogens in mammalian hosts. Such pathogens present a significant risk for the emergence of new infectious diseases in humans. This genomic variant is within the gene TNIK, which is a germinal center kinase (GCK). GCKs are participants in both adaptive and innate immune regulation. However, the function of TNIK is not fully understood. We quantify the adaptive force on the C allele due to the pathogens as 0.04 GEU’s/viral species.

MAP4K2 Silencing Overcomes IMiDs-Resistance in Multiple Myeloma

Recently, mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2) has emerged as an important key regulator of the stress-activated MAPK core signaling pathways. MAP4K2, also called Germinal Center Kinase (GCK), is predominantly and highly expressed in the germinal center of B cells. Recently we have shown that MAP4K2 knockdown in K- or N-RAS mutated MM cells induces MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins (Li et al. Blood 2021). Importantly, MAP4K2 silencing induces IKZF1 protein degradation without affecting IKZF1 mRNA level. Furthermore, IMiDs-resistant K-RAS Mut MM cells are sensitive to MAP4K2 inhibition induced IKZF1 degradation and cell growth suppression, suggesting that MAP4K2 inhibition overcomes IMiDs resistances in MM.To further validate MAP4K2 inhibition as a novel strategy to overcome IMiDs-resistance, we generated lenalidomide-resistant human myeloma cell lines. In this model, MM1S-LEN RES cells showed significantly decreased expression of CRBN protein compared to the parent cells. Accordingly, upon lenalidomide treatment, CRBN-mediated down-regulation of IKZF1, c-MYC, IKZF3 and IRF4 were abrogated in the MM1S-LEN RES cells. As expected, MM1S-LEN RES cells were resistant to lenalidomide induced growth inhibition in cell proliferation assay. In contrast, MAP4K2 inhibition using TL4-12 potently induced IKZF1, c-MYC, and IRF4 downregulation as well as cell proliferation inhibition, demonstrating that MAP4K2 regulates IKZF1 and cell growth independently of CRBN. These results indicate that MAP4K2 is a novel therapeutic target to overcome IMiDs-resistance MM.Iberdomide (CC-220) is an orally available IMiD® compound under development for the treatment of relapsed/refractory multiple myeloma. Previous biochemical and structural studies demonstrated that Iberdomide binds to cereblon with a higher affinity than lenalidomide or pomalidomide. Here, we evaluated the combination effects of Iberdomide with MAP4K2 silencing in MM. Tet-on sh-MAP4K2 lentivirus were introduced into RAS Mut MM cells to establish the inducible MAP4K2 knockdown cells upon doxycycline treatment. To address the combination effects, Tet-on sh-MAP4K2 RAS Mut MM cells were treated with doxycycline and different dosages of iberdomide. We found that MAP4K2 silencing strongly increased iberdomide-induced apoptosis (iberdomide alone vs. with MAP4K2 KD: 32% vs 92). Similar, in western blot assays, MAP4K2 silencing combined with Iberdomide significantly enhanced downregulation of IKZF1, c-MYC, and IRF4 compared to the iberdomide treatment alone. These data suggest that combination of iberdomide and MAP4K2 inhibition have synergetic anti-MM effects.Taken together, our findings demonstrate that MAP4K2 is a novel therapeutic target to bypass IMiDs resistance in RAS mutated MM. Combination of MAP4K2 inhibition with Iberdomide results in synergetic anti-cancer effects in MM, therefore could be a potent novel therapeutic regimen for patients with relapsed/refractory multiple myeloma. DisclosuresMarcireau: Sanofi: Current Employment.

OAB-032: Targeting GCK in RAS-mutant multiple myeloma offer a promising therapeutic approach

<h3>Background</h3> Next generation sequencing revealed frequent mutations of NRAS, KRAS or BRAF in up to 50% of newly diagnosed multiple myeloma (MM) patients. <h3>Methods</h3> Specific K- or N-RAS knockdowns led to strong decrease in MM cell viability if harbored the respective oncogenic isoform, but minimal effects were observed if only the wild-type isoform was present, presenting RAS signaling as the key driving oncogenes in the mutation bearing patients. Unfortunately, no inhibitor for RAS mutation is available for clinical use in MM. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK) is an upstream activator in the MAPK pathway. <h3>Results</h3> Our data showed that GCK (MAP4K2) knockdown in MM cells induced MM cell growth inhibition, associated with the downregulation of critical transcriptional factors including IKZF1/3, BCL-6, and c-MYC proteins. Moreover, GCK silencing only led to significantly decreased GCK mRNA, however, did not affect IKZF1 expressions at mRNA level. We next tested the effects of GCK inhibitor TL4-12 on MM. Given the fact that RASMut MM cells have higher GCK expression level and are more sensitive to GCK knockdown, we compared mutated RAS MM cells with wild-type RAS MM cells. TL4-12 significantly inhibited the growth of mutated RAS MM cells, with IC50 5-10 fold lower compared to wild-type RAS MM cell lines. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Our data showed that GCK knockdown also downregulates IKZF1 protein level, indicating that IKZF1 is under the regulation of GCK. Concomitant treatment with proteasome inhibitor PS-341 blocked TL4-12 induced IKZF1 downregulation, suggesting that inhibition of GCK induces IKZF1 protein degradation. However, since IMiDs-resistant RPMI-8226 and JJN3 MM cells are sensitive to GCK inhibition, we hypothesized that the IKZF1 degradation mechanism induced by GCK inhibition is different from IMiDs and independent of CRBN. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells and examined the response to IMiDs and GCK inhibitor. CRBN silencing in H929 cells resulted in lenalidomide resistance. In contrast, CRBN silencing failed to rescue H929 MM from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. <h3>Conclusions</h3> Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibition downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance.

TNIK influence the effects of antipsychotics on Wnt/β-catenin signaling pathway.

RationaleTraf2- and Nck-interacting kinase (TNIK), a member of germinal center kinase (GCK) family, has been implicated as a risk factor in schizophrenia and bipolar disorder as well as the action of antipsychotics. TNIK is an essential activator of Wnt/β-catenin signaling pathway which has been identified involved in the mechanism underlying the effects of antipsychotics. Thus, the effects of TNIK on antipsychotics may be achieved by influencing Wnt/β-catenin signaling pathway proteins.Objectives and methodsIn the current study, the effects of up- or downregulated TNIK on β-catenin, T-cell factor 4 (TCF-4), glycogen synthase kinase-3β (GSK3β), and phosphorylated GSK3β (p-GSK3β) were examined in the human glioma U251 cells. Then, we observed the effects of antipsychotics (clozapine and risperidone) on the above proteins and evaluated the role of differentially expressed TNIK on antipsychotic-treated cell groups.ResultsThe result showed that clozapine treatment decreased β-catenin and TCF-4 levels in U251 cells, and risperidone had the similar effects on β-catenin and p-GSK3β. The downregulated TNIK using siRNA impeded the regulation of antipsychotics on Wnt pathway proteins via increasing the expression levels of TCF-4, β-catenin, or p-GSK3β, whereas the upregulated TNIK made no significant change.ConclusionsThe influence of TNIK on the effects of antipsychotics may be partly through Wnt/β-catenin signaling pathway.

Germinal Center Kinase As a Therapeutic Target in Multiple Myeloma

Introduction: Despite many new drug approvals, Multiple Myeloma remains an incurable disease due to development of drug resistance. This process is driven by clonal evolution in which kinases play a major role. Germinal center kinase (GCK) is the founding member of the GCKs, a group of Ser/Thr kinases homologous to the saccharomyces cerevisiae Ste20p, and participates in B cell differentiation. Among human tissues, GCK is highly expressed in transformed lymphocytes, spleen and whole blood. However, the expression and function of GCK in human myeloma is totally unknown.Methods and Results: First we analyzed the expression level of GCK in human MM cells and found that GCK is abundantly expressed in all the MM cell lines we tested. To determine whether GCK is required for MM cell growth, we introduced inducible shRNA to knock down GCK expression in MM cells by lentiviral mediated transduction. MM cell lines MM.1S and RPMI-8266 were stably transduced with shRNA lentivirus generated from a robust inducible knockdown vector pLKO-tet-On. Doxycycline-induced GCK-tet-on-shRNA expression resulted in GCK protein knockdown and significant inhibition of cell growth by 75% in WST-1 proliferation assay. In accordance, cell cycle analysis revealed that the knockdown of GCK decreased cells in S-phase with concomitant increase in G0/G1 growth arrest (55% vs 67%). Importantly, GCK knockdown resulted in significant increase (3.21% vs 60.8%) of apoptotic cells suggesting that inhibition of GCK is a potential therapeutic target in MM cells. This was further confirmed by treating MM cells lines (MM.1S, RPMI-8266 and H929) with GCK inhibitors TL4-12, NG25 and Bay 61-3606. We found that these drugs dose dependently and significantly inhibit multiple myeloma cell growth, with IC50 7, 1.6 and 0.5 µM, respectively in H929 cells by WST-1 proliferation assay. Mechanistic studies showed that Bay 61-3606 dose-dependently decreases the protein levels of IKZF1/IKZF3 and c-Myc, increases p53 level and induces PARP cleavage resulting in cell cycle G1 arrest (60.8 vs. 69.8 with 1 µM) and apoptosis (9.16 vs. 29.4 at 1 µM) in MM.1S cells. To confirm the critical role of GCK in in vivo growth of MM cells, we generated subcutaneous MM xenografts in SCID Beige (CB17.Cg-PrkdcscidLystbg-J/Crl) mice using doxycycline-inducible GCK shRNA MM.1S cells. Doxycycline or vehicle treatment was started after the tumor was established on day 16. In contrast to vehicle-treated MM.1S-tet-on-shGCK or doxycycline-treated MM.1S-tet-on-sh-control tumors, doxycycline treated animals bearing MM.1S-tet-on-shGCK xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 30 days. Immunohistochemistry staining of tumors confirmed the decrease of GCK and IKZF1 expression in doxycycline-treated mice bearing MM.1S-tet-on-shGCK tumors compared with tumors of vehicle- or non-doxycycline treated mice.Conclusion: Our findings show that GCK is a potential target for multiple myeloma therapy. GCK inhibition results in significant anti-MM effects by inducing cell cycle arrest and cell apoptosis accompanied by IKZF1/3 down regulation. Furthermore, the inhibition of in vivo tumor growth correlates with the doxycycline-induced GCK knockdown, further confirming the critical role of GCK in MM tumorigenesis. DisclosuresLentzsch:Amgen: Consultancy; BMS: Consultancy; Caelum Biosciences: Other: leadership position and stock.

The Traf2 and Nck-Interacting Kinase Is a Conserved Regulator of Self-Renewal in Hematopoietic and Leukemia Stem Cells

The Traf2 and Nck-interacting kinase (TNIK) is a member of the germinal center kinase (GCK) family. We have recently found that signaling through the tumor necrosis factor superfamily receptor CD27 activates Wnt target genes through Traf2-TNIK-β-catenin signaling, promoting leukemia development and progression in acute and chronic myeloid leukemia (AML and CML).To explore how TNIK signaling regulates hematopoietic and leukemia stem cells (HSCs and LSCs), we generated a constitutive TNIK-/- knockout mouse.Under steady-state conditions, total bone marrow (BM) cellularity of naïve TNIK-/- mice was slightly reduced (mean BM count (x108): TNIK-/- = 1.42; TNIKwt/wt = 2.15, p < 0.0132). In contrast, no differences in absolute number of lineage negative (Lin-) cells, as well as the more primitive subset Lin-; Sca1+; c-kit+ (LSK) cells could be observed. Similarly, proportions of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs) and multipotent progenitors (MPPs) did not differ between TNIK-/- and TNIKwt/wt mice. Functionally, naïve TNIK-/- and TNIKwt/wt HSCs displayed in vitro a comparable serial colony formation capacity in methylcellulose. Altogether these data suggest that TNIK does not affect the maintenance and self-renewal of HSCs under steady-state conditions.Next the impact of TNIK on hematopoietic recovery after genotoxic stress was analyzed in vivo. TNIK-/- and TNIKwt/wt mice were injected i.p. with 5-Fluorouracil (5-FU) (150mg/g bodyweight) and BM cellularity as well as HSC phenotype and function were assessed 9 days post injection. The total BM cellularity was significantly higher in TNIK-/- mice compared to controls (mean BM count (x106/ml): TNIK-/- =10.25; TNIKwt/wt =4.55, p< 0.0382). Furthermore, LSKs were significantly reduced in frequency in TNIK-/- mice (% LSK of whole BM: TNIK-/- = 2.93; TNIKwt/wt = 7.073, p < 0.0009) but not in absolute numbers. Interestingly, however, the total number LT-HSCs was significantly reduced in the BM of TNIK-/- mice (mean LT-HSC count (x103): TNIK-/- = 5.628; TNIKwt/wt = 16.725, p < 0.0101). These results were functionally confirmed in vivo by competitive BM transplantation (BMTx) of whole BM from 5-FU treated mice into lethally irradiated secondary recipients. Mice receiving BM from 5-FU-treated TNIK-/- mice showed a significantly reduced peripheral engraftment capacity 6 weeks post BMTx compared to controls (mean peripheral blood chimerism: TNIK-/- = 64.6%; TNIKwt/wt = 35.4%, p < 0.0001), suggesting that TNIK signaling mediates HSC maintenance during genotoxic stress conditions in vivo.In parallel the role of TNIK signaling was analyzed in myeloid LSCs. CML-like disease was induced in mice by transplanting pMSCV-BCR-ABL1-IRES-GFP transduced TNIK-/- or TNIKwt/wt LSKs into BL/6 mice. Even though CML developed similarly as indicated by measuring BCR-ABL1-GFP+ granulocytes in the peripheral blood, primary TNIK-/- CML mice survived significantly longer compared to TNIKwt/wt CML mice (Median survival (days): TNIK-/- = 33, TNIKwt/wt = 23.5, p < 0.0216) with 2 out of 7 mice surviving long-term. Next, we determined the effect of TNIK signaling on the functionality of FACS sorted Lin-; GFP+; Sca1+; c-kit+ LSC in vitro in serial colony forming assays and in vivo in secondary transplantations. Serial colony formation capacity of TNIK-/- CML LSCs was significantly reduced after re-plating in vitro (CFUs fold change (TNIK-/- vs TNIKwt/wt): 1st Plating = 0.89, P<0.5149, 2nd Plating = 0.42, 9 < 0.0002). In line with these findings, only 1 out of 9 mice secondarily transplanted with LSCs from primary TNIK-/- CML mice developed CML within 90 days, while all 9 recipients receiving TNIKwt/wt LSCs succumbed to the disease within 23 days after transplantation (p < 0.0001). These results suggest that TNIK signaling also promotes self-renewal of CML LSCs.To summarize, we have described for the first time TNIK signaling as a commonly conserved regulator in stress-induced hematopoiesis and CML, which promotes stem cell self-renewal and maintenance. As TNIK inhibitors are currently developed for the treatment of different kinds of solid tumors, TNIK may be an attractive therapeutic target for LSCs in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Effects of weekly selinexor, bortezomib, dexamethasone (XVd) versus standard twice weekly bortezomib and dexamethasone (Vd) on RAS-mutated previously treated multiple myeloma (MM).

8027 Background: Activating mutations of the RAS genes NRAS, KRAS, and HRAS ( RASmut) occur in up to 50% of MM and portend poor survival and high recurrence rates. MM cells with RASmut are susceptible to inhibition of germinal center kinase (GCK), resulting in IKAROS degradation independent of cereblon (CRBN). Selinexor is a selective inhibitor of nuclear export (SINE) compound that can induce IKAROS degradation through CRBN-independent pathways to overcome immunomodulatory drug resistance. We explored the benefit of selinexor treatment for pts with RASmut MM. Methods: In the randomized BOSTON study, pts with MM after 1-3 therapies received weekly XVd or twice weekly Vd. In the single-arm STORM study pts with penta-treated, triple class refractory MM were treated with twice weekly Xd. Both treatment regimens are now FDA approved. Mutations were assessed post-hoc by exome sequencing of 119 and 52 pts from BOSTON and STORM, respectively. Pts were considered RASmut if their MM had NRAS, KRAS or HRAS mutations in codons 12, 13 or 61. Results: There were 54 pts (45%) with RASmut in BOSTON (XVd = 26, Vd = 28), and 17 (33%) in STORM. In BOSTON, pts with RASmut MM treated with XVd had significantly longer progression-free survival (PFS) than those treated with Vd (median [med] = 12.9 vs 6.7 months [mo], hazard ratio [HR] = 0.48 [95% CI 0.24-0.97], p = 0.039). For pts treated with Vd, those with RASmut had significantly shorter overall survival (OS) compared to RASwild-type (WT) (med = 16.8 mo vs not reached [NR], HR = 2.87 [95% CI 1.03-7.99], p = 0.035). PFS was not significantly different (med = 6.74 vs 9.82 mo, HR = 1.64 [95% CI 0.88-3.07], p = 0.122). Amongst pts on XVd, there was no difference in survival between RASmut and RASWT pts (PFS: med = 12.8 vs 12.9 mo, HR = 1.08 [95% CI 0.52-2.26], p = 0.83; OS: med = NR vs NR, HR = 0.94 [95% CI 0.36-2.45], p = 0.91). In STORM, pts with RASmut had shorter OS compared to RASwt pts (med = 6.1 vs NR, HR = 2.05 [95% CI 1.22-5.19], p = 0.010). Preliminary studies to explore the mechanisms of action related to RASmut MM sensitivity to XVd demonstrated that selinexor treatment in vitro leads to downregulation of GCK. Conclusions: Despite typically having the worst outcomes, pts with RASmut MM had a similar benefit from XVd as RASWT MM, showing that the XVd combination can overcome RASmut. Mechanistically, selinexor induced down regulation of GCK and enhanced killing of RASmut MM cells. With a manageable safety profile, the XVd regimen could provide a viable treatment option to improve survival of pts with MM with RAS mutations. Clinical trial information: NCT03110562. [Table: see text]

Targeting the GCK pathway: a novel and selective therapeutic strategy against RAS-mutated multiple myeloma

In multiple myeloma (MM), frequent mutations of NRAS, KRAS, or BRAF are found in up to 50% of newly diagnosed patients. The majority of the NRAS, KRAS, and BRAF mutations occur in hotspots causing constitutive activation of the corresponding proteins. Thus, targeting RAS mutation in MM will increase therapeutic efficiency and potentially overcome drug resistance. We identified germinal center kinase (GCK) as a novel therapeutic target in MM with RAS mutation. GCK knockdown (KD) in MM cells demonstrated in vitro and in vivo that silencing of GCK induces MM cell growth inhibition, associated with blocked MKK4/7-JNK phosphorylation and impaired degradation of IKZF1/3, BCL-6, and c-MYC. These effects were rescued by overexpression of a short hairpin RNA (shRNA)-resistant GCK, thereby excluding the potential off-target effects of GCK KD. In contrast, overexpression of shRNA-resistant GCK kinase-dead mutant (K45A) inhibited MM cell proliferation and failed to rescue the effects of GCK KD on MM growth inhibition, indicating that GCK kinase activity is critical for regulating MM cell proliferation and survival. Importantly, the higher sensitivity to GCK KD in RASMut cells suggests that targeting GCK is effective in MM, which harbors RAS mutations. In accordance with the effects of GCK KD, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 and led to MM cell proliferation inhibition accompanied by induction of apoptosis. Here, our data identify GCK as a novel target in RASMut MM cells, providing a rationale to treat RAS mutations in MM. Furthermore, GCK inhibitors might represent an alternative therapy to overcome immunomodulatory drug resistance in MM.

Gck Inhibition Is a Novel Therapeutic Strategy for RAS Mutated Multiple Myeloma and Overcomes Resistance to IMiDs

Introduction: RAS oncogenes are the most frequently mutated gene family in human cancers. 50% of newly diagnosed multiple myeloma patients carry a RAS/MAPK pathway mutation, with a rising percentage in the relapsed situation1. Thus, targeting RAS mutations in multiple myeloma will increase therapeutic efficiency and potentially overcome drug-resistance. Unfortunately, RAS mutations have been considered "undruggable" due to a lack of traditional small molecule binding pockets on the proteins. Therefore, key component in the RAS/MAPK pathway may represent an alternative therapeutic target for MM. Germinal center kinase (GCK), also named mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2), is an upstream activator in the MAPK pathway. Indeed, we recently discovered the critical role of GCK in RAS mutated (RASmut) MM cell survival and growth. GCK knockdown in RASmut MM cells induced MM cell growth inhibition both in vitro and in vivo. However, the detailed mechanism is yet to be defined. Methods and Results: Our previous data showed that GCK knockdown induces MM cell growth inhibition, associated with the blockage of MKK4/7-JNK phosphorylation and the downregulation of critical transcriptional factors (TFs) including IKZF1/3, BCL-6, and c-MYC proteins. To confirm that GCK knockdown downregulates IKZF1/3 etc at protein level but not mRNA level, we conducted real-time PCR on GCK knockdown MM cells and compared the expression of GCK and TFs to the empty vector (EV) infected MM cells. Results showed that shRNA induced GCK silencing only led to the significantly decreased GCK mRNA, however, did not affect IKZF1 and c-MYC expressions at mRNA level. Consistent with the effects of GCK knockdown, the GCK inhibitor TL4-12 dose-dependently downregulated IKZF1 and BCL-6 proteins, inhibited MM cell proliferation and induced cell apoptosis. IKZF1/3 are the key targets of the immunomodulatory drugs (IMiDs), which are the backbone of MM therapy. IMiDs bind to cereblon (CRBN) and induce IKZF1/3 protein degradation, which subsequently lead to MM cell growth inhibition. Importantly, our data showed that IMiDs-resistant RPMI-8226 MM cells have high expression of GCK. GCK knockdown and inhibition induced IKZF1 downregulation, triggered growth inhibition and cell apoptosis in RPMI-8226 cells, suggesting that GCK regulates IKZF1 degradation via a CRBN-independent mechanism. To confirm this hypothesis, we silenced CRBN in N-Rasmut H929 MM cells by shRNA lentiviral infection and examined the response to IMiDs and GCK inhibitor. CRBN knockdown was confirmed by western blotting. CRBN silencing in H929 cells resulted in lenalidomide (LEN) resistance, evidenced by the WTS proliferation assay. In contrast, CRBN silencing failed to rescue N-Rasmut H929 MM cells from TL4-12 induced proliferation inhibition and IKZF1 downregulation, confirming that GCK regulated IKZF1 and cell growth is independent of CRBN. Conclusion: Taken together, our data demonstrated that GCK inhibition induces cell growth inhibition and triggers apoptosis especially in RASmut MM cells. Importantly, GCK inhibitor downregulates IKZF1 via a CRBN-independent mechanism. Our findings thus provide a rationale for the clinical evaluation of targeting GCK in RASmut MM patients and further mechanistic insight into the role of GCK in MM tumorigenesis as well as drug resistance. GCK inhibitors may represent a novel therapy for the treatment of RASmut MM patients, especially those who are resistant to IMiDs as well as with refractory or relapsed MM. References Walker, B.A., et al. Mutational Spectrum, Copy Number Changes, and Outcome: Results of a Sequencing Study of Patients With Newly Diagnosed Myeloma. J Clin Oncol33, 3911-3920 (2015). Disclosures Marcireau: Sanofi: Current Employment. Lentzsch:Karyopharm: Research Funding; Mesoblast: Divested equity in a private or publicly-traded company in the past 24 months; Janssen: Consultancy; Sorrento: Consultancy; Caelum Biosciences: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Celularity: Consultancy; Magenta: Current equity holder in private company; Sanofi: Research Funding.

Enhanced anti-tumor response upon deficiency of Msn kinase MINK1 in mice

Abstract Misshapen (Msn)/NIK-related kinase 1 (MINK1) is a serine/threonine kinase belongs to the germinal center kinase (GCK) family. Previous studies indicated the correlation of MINK1 with tumor cell senescence and Th17 differentiation. Its dual role in modulating both tumor growth and T cell function indicated MINK1 a possible intervention target in tumor immunotherapy. We have utilized the MINK1 deficient mice to explore the possible role of MINK1 in the anti-tumor immune response. By using the subcutaneous tumor-xenograft model, we have found that MINK1 deficiency in mice resulted in slower tumor growth. We have also accumulated evidence in adoptive transfer and CD8+ T cells depletion experiments to show that the genetic ablation of MINK1 suppressed tumor growth primarily via CD8+ T cells without affecting other immune cells. Along with which, MINK1 deficient CD8+ T cells are superior in both tumor infiltration and the expression of effector markers such as TNF-α, IFN-γ, and Granzyme-B when compared to WT CD8+ T cells. Lower expression of exhaustion markers such as PD-1, TIM-3, and TIGIT were also observed in MINK1 KO CD8+ T cells. We are currently exploring the possible mechanisms of how MINK1 regulated CD8+ T cell function, and examine the possibility to target MINK1 in experimental tumor immunotherapy.

Activity of the multikinase inhibitor TP‐0903 in RAS mutant acute myeloid leukemia

Clonal heterogeneity and co‐occurring mutations contribute to drug resistance in AML. Leukemia samples from patients progressing on gilteritinib, a FLT3 inhibitor, were selected for RAS mutant clones, which are known to contribute preclinically to resistance (McMahon CM, Cancer Discov 2019). Previously, we demonstrated that TP‐0930, a multi‐kinase inhibitor, targets FLT3‐ITD and resistance‐conferring tyrosine kinase domain mutations in preclinical models. Here, we present the activity of TP‐0903 in RAS pathway mutant models of AML.KiNativ assay was performed in OCI‐AML3 cells (NRAS Q61L) treated with TP‐0903 (100 nM, 2h). Inhibition of cell viability was evaluated in AML cell lines (MTT) and primary samples (CellTiter Glo). Inhibition of AURKA/B, AKT, ERK, MCL‐1, and BCL‐2 were determined by western blot. Apoptosis, cell cycle, and differentiation assays were performed in OCI‐AML3 using annexin V, DAPI and CD11b‐APC staining. In vivo activity of TP‐0903 was evaluated in a systemic OCI‐AML3‐Luc+ xenograft model; 5e5 luciferase expressing cells were tail vein injected to NSG mice and TP‐0903 50 mg/kg or vehicle was given orally once dailyx5/week. Progression of leukemia was monitored via bioluminescence imaging and Kaplan Meier was used for survival analysis.In Kinativ assay, inhibition of native kinases were similar to those observed in a previous binding assay. We observed that TP‐0903 inhibited ACK1 (activated CDC42 kinase 1 or TNK2) and GCK (germinal center kinase or MAP4K2) by 87 and 46%, respectively. Inhibition of ACK1 (Kd = 7.3 nM, IC50 = 30.7 nM) and GCK (Kd = 1.8 nM, IC50 = 2.5 nM) was confirmed in binding and kinase assays. In RAS mutant AML cell lines (OCI‐AML3, THP1, HL60, and U937), TP‐0903 inhibited viability with IC50 values of 26 – 99 nM. In OCI‐AML3, TP‐0903 at 100 nM inhibited pAURKA/B, pAKT and MCL‐1; caused a G2‐M arrest and polyploidy (20 nM, 24h); and induced apoptosis (20–50 nM, 72h). TP‐0903, compared to gilteritinib, was more potent in inhibiting 4 ex vivo human primary AML samples with N/KRAS mutations co‐occurring with wt FLT3 or FLT3‐ITD, along with other mutations (IC50 range, 21 – 60 vs 260 – 1370 nM). In a 14‐day CFU assay, TP‐0903 inhibited colony formation of a FLT3‐ITD and NRAS mutant primary sample compared to DMSO (mean, 65 vs 188 CFUs). In an OCI‐AML3‐Luc+ xenograft study, TP‐0903 suppressed the outgrowth of leukemia at day 21 (P&lt;0.0001) and prolonged median survival by 9 days compared to veh‐treated mice (P&lt;0.0001). At study endpoint, spleen weight was higher in veh‐treated mice than in TP‐0903‐treated mice (mean ± SD, 333 ± 23 vs 181 ± 21 mg, P=0.0029).TP‐0903, a novel multi‐kinase inhibitor, shows promising in vitro and in vivo activity in RAS mutant AML. In a previous report (Nonami A, Blood 2015), an integrated approach involving cell‐based pharmacologic screening combined with KiNativ, gene expression profiling and mechanism studies, identified 2 kinase signaling pathways (ACK1/AKT and GCK) that synergistically contributed to the growth of NRAS mutant leukemia cells, including OCI‐AML3. Based on our data, TP‐0903 may exert its activity in RAS mutant AML via inhibiting ACK1 and GCK.