Role of protein kinases CK1α and CK2 in multiple myeloma: regulation of pivotal survival and stress-managing pathways
Multiple myeloma (MM) is a malignant tumor of transformed plasma cells. MM pathogenesis is a multistep process. This cancer can occur de novo (rarely) or it can develop from monoclonal gammopathy of undetermined significance (most of the cases). MM can be asymptomatic (smoldering myeloma) or clinically active. Malignant plasma cells exploit intrinsic and extrinsic bone marrow microenvironment-derived growth signals. Upregulation of stress-coping pathways is also instrumental to maintain MM cell growth. The phylogenetically related Ser/Thr kinases CSNK1A1 (CK1α) and CSNK2 (CK2) have recently gained a growing importance in hematologic malignancies arising both from precursors and from mature blood cells. In multiple myeloma, CK1α or CK2 sustain oncogenic cascades, such as the PI3K/AKT, JAK/STAT, and NF-κB, as well as propel stress-related signaling that help in coping with different noxae. Data also suggest that these kinases modulate the delivery of growth factors and cytokines from the bone marrow stroma. The “non-oncogene addiction” phenotype generated by the increased activity of CK1α and CK2 in multiple myeloma contributes to malignant plasma cell proliferation and survival and represents an Achilles’ heel for the activity of small ATP competitive CK1α or CK2 inhibitors.
KeywordsMultiple myeloma CK1 CK2 Bone marrow microenvironment Survival signaling pathways Non-oncogene addiction
Acute myeloid leukemia
Ataxia telangiectasia and Rad3-related protein
Bone marrow stromal cells
Bruton tyrosine kinase
Chronic lymphocytic leukemia
Chronic myeloid leukemia
Diffuse large B cell lymphoma
Extracellular signal-regulated kinase
Fibroblast growth factor 3
Glycogen synthase kinase 3
Hsp70-Hsp90 organizing protein
Insulin-like growth factor-I
Interferon regulatory factor 4
Mantle cell lymphoma
Mesenchymal stromal cells
Mammalian target of rapamycin complex 1
Nuclear factor kappa-light-chain-enhancer of activated B cells
Phosphatase and tensin homolog
Signal transducer and activator of transcription 3
Telomere maintenance 2
Tumor necrosis factor-α
Tel2 interacting protein 1
Unfolded protein response
Multiple myeloma (MM) is the second most frequent hematologic malignancy, accounting for about 13% of all blood cancers . The bulk of the tumor is represented by transformed plasma cells (PCs), which accumulate firstly in the bone marrow (BM) in a microenvironmental niche that supports their growth . Along with the disease progression, malignant PCs lose their dependency from the BM and gain features of autonomous growth with widespread dissemination of plasma cell leukemia [3, 4]. Clinically, MM is characterized by target organ damage, with anemia, renal insufficiency and bone resorption/loss with bone pain, hypercalcemia, and pathological fractures. In addition, MM patients develop immune-paralysis with hypogammaglobulinemia and susceptibility to infections .
The molecular and genetic features of MM have lately been described. MM are divided in cases bearing chromosomal translocations affecting the IgH locus (30%); cases with hyperdiploidy (trisomies) of odd chromosomes 3, 5, 7, 9, 11, 15, 17, and 19 (40–45%); cases with both the alterations (15%); and cases with other abnormalities (10–15%) [6, 7]. The genomic analysis of MM cases has revealed a complex genetic architecture that suggests a continuous clonal evolution in a Darwinian process and few recurrent mutations concentrated in clusters of genes, which regulate, among others, the translation process, chromatin modification and gene transcription, including the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway .
Besides intrinsic alterations in MM PCs, an aberrant BM microenvironment participates in MM pathogenesis. The stromal niche surrounding malignant PCs is able to deliver trophic signals represented by cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), growth factors, such as insulin-like growth factor-I (IGF-I) and related proteins, and soluble glycoproteins, such as Wnt and Hedgehog. All these signaling cascades inside MM cells shift the milieu towards osteoclast propelling features and promote aberrant neoangiogenesis [2, 8]. Therefore, MM cells and bone marrow stromal cells (BMSCs) depend on a number of signaling cascades whose regulation is still largely unknown. MM cells rely also on intracellular pathways that have the ability to manage a different array of stresses, including the proteotoxic, replicative, and oxidative stress [9, 10, 11]. Thus, molecules acting as “stress managers” may become essential for the optimal fitness of malignant PCs. Examples are the transcription factor IRF4, which is part of a rewired transcriptional program in malignant PCs as compared to normal counterparts , the kinase ATR , and the scavenger enzyme SOD2 .
Mutations as well as hyper-function of certain fundamental proteins and cascades may cause tumor promotion and progression. In this context, it is not doubtable that native or newly generated protein kinases (PK) may become pivotal players. As a proof of concept that PK may be central in oncogenesis is the clear evidence of the lethal consequences for many tumor types caused by their inhibition. Some examples are the drugs imatinib, gefitinib, ibrutinib, or fostamatinib, which target a number of receptor/cytosolic tyrosine kinases and have proven to be clinically effective therapeutic options for solid tumors, chronic myeloid leukemia (CML) or chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphomas. However, despite the fact that in several B cell malignancies protein kinases represent valid therapeutic targets, this proof of principle is lacking in MM.
In this regard, among protein kinases driving MM cell survival, recently, the Ser/Thr kinases CK1α and CK2 have been shown to play an important role as regulators of signal transduction and stress response [15, 16, 17]. We will herein review CK1α and CK2 function in MM and discuss the potential of targeting their kinase activity as a suitable therapeutic strategy for this B cell-derived tumor.
Protein kinase CK1α: emerging roles in cancer
CK1α belongs to a family of highly conserved monomeric Ser/Thr kinases composed by seven members encoded by different genes (α, β, γ1, γ2, γ3, δ, and ε), displaying the highest homology in their kinase domains (50–90% identical) with similar substrate specificity. CK1 members regulate membrane biology, molecular transport, signal transduction, transcription, translation, and DNA damage response [18, 19]. In the last few years, CK1α, encoded by the CSNK1A1 gene, has been involved in cancer with a role that seems multifaceted.
CK1α inhibition, leading to stabilization of β-catenin, acts as a tumor promoter in the absence of p53 in intestinal epithelial cells, while its inactivation does not turn into tumor formation as long as p53 is active [20, 21]. Nevertheless, CK1α is a tumor promoter in acute myeloid leukemia (AML), provided there is an intact p53 . CK1α has been shown to negatively regulate Ras-induced autophagy in models of Ras-driven transformation by controlling the phosphorylation of FOXO3A on S318/321 and its subsequent nuclear extrusion .
Other reports have involved CK1α in tumors. CK1α is a tumor supporter in diffuse large B cell lymphoma (DLBCL) of activated B cell subtype, inducing the activation of NF-κB through the regulation of the CBM1 complex (CARD11, BCL10, MALT1) . Mutations of CK1α have been detected in melanoma, clear cell renal cell carcinoma [25, 26], colon cancer , esophageal adenocarcinoma , adult T cell leukemia/lymphoma , and del(5q) myelodisplatic syndromes (MDS) . Schneider and colleagues demonstrated a prominent role of CK1α in del(5q) MDS. Our group and others have demonstrated an oncogenic role of CK1α in MM [17, 31].
Altogether, a growing body of data points to a potential role for CK1α in carcinogenesis in different tumor types.
Protein kinase CK2: an indispensable molecule for cancer cell survival
Protein kinase CK2 is a Ser/Thr kinase that regulates critical cellular processes. It is composed by a tetramer of two catalytic (α or α’) and two regulatory subunits (β) . The substrate specificity is believed to rely on the β subunit even though tetramer-independent functions of the α and β moieties have been recognized. CK2 has been involved in cell proliferation, apoptosis, transcription and translation, adhesion and motility, and stress-coping [33, 34, 35]. CK2 has hundreds of substrates , among which are pivotal oncogenic proteins, like AKT [37, 38], c-Myc , NF-κB [8, 40], and signal transducer and activator of transcription 3 (STAT3) . Given its multilateral involvement in cell biology, it is not surprising that high CK2 activity and expression have been correlated to poor prognosis and resistance to anti-cancer agents in different types of cancers, including breast , lung , prostate , renal , bladder carcinoma , melanoma , and hematological malignancies . CK2 acts as a typical “non-oncogene addiction” molecule, since cancer cells strongly rely on it even though it is generally not altered by typical mutations leading to a gain of function phenotype [47, 48]. However, recently, CK2β deletions were identified in 15% of cases of early relapsing DLBCL patients compared to those of late relapsing .
The mechanisms by which CK2 sustains malignant tumor growth are numerous. CK2 promotes cancer cell survival by activating tumor-promoting oncogenes (such as c-Myc  in lymphoma, Ras-ERK  in melanoma , AKT [51, 52, 53, 54] in bladder, colon, and blood cancers) or by inhibiting tumor suppressors (such as PTEN) [53, 55].
CK2 also strongly supports the activity of important signaling cascades, such as the NF-κB, in breast cancer  as well as in multiple myeloma, lymphoma  and leukemia , Wnt-β-catenin, , Hedgehog in pleural mesothelioma , and STAT3-dependent signaling in solid and in hematological tumors . CK2 phosphorylates NF-κB RelA/p65 on Ser529, increasing p65 transcriptional activity downstream external stimuli . CK2 positively modulates STATs by phosphorylating Ser727 of STAT3 . This kinase regulates β-catenin stability [60, 61] and Gli1 function . Besides its role as a signaling regulator, CK2 is involved in cellular processes of stress-coping/fitness augmentation. For instance, CK2 regulates the DNA damage response [63, 64], autophagy , and endoplasmic reticulum (ER) stress [15, 66]. A major role in cell survival is believed to rely on CK2 modulation of caspase activity [67, 68, 69].
CK1α and CK2 in multiple myeloma: regulation of signal transduction
Others and our group demonstrated that CK1α is a pro-growth kinase in MM. In the work by Hu et al. , CK1α was found to promote survival and proliferation of MM cell lines and cMyc/KRasV12-transduced BaF3 cells in xenograft mouse models. CK1α inhibition led to higher interferon-α and TNF-α signaling. More recently, we demonstrated that CK1α is highly expressed in the vast majority of MM patient PCs (in a large microarray data set series) compared to that in healthy PCs . CK1α loss of function with a pan-CK1 inhibitor (D4476) or RNA interference (RNAi) was accompanied to MM cell apoptosis, cell cycle arrest and downregulation of β-catenin, and AKT survival signaling, in a mechanism that could involve caspase and p53 . Of note, CK1α inactivation was able to overcome BMSCs protection. Moreover, CK1α inhibition synergically boosted bortezomib and lenalidomide cytotoxicity. Interestingly, lenalidomide treatment of MM cells determined a deregulation of CK1α expression, in a time- and dose-dependent manner, in a mechanism similar to that observed in other cell types .
CK2 is overexpressed and enzymatically more active in malignant MM PCs from patients and cell lines compared to that in healthy controls . CK2 was found localized in the cytoplasm, in the nucleus, and in a small fraction also in the ER of malignant PCs . The use of ATP competitive CK2-specific inhibitors like tBB, K27, and the newly developed clinically graded CX-4945 (silmitasertib) caused malignant PCs apoptosis, being less toxic to the non-malignant counterparts [8, 15, 16, 71]. From a molecular standpoint, CK2 inactivation with chemicals or by RNAi in MM impacted on two main signaling pathways, the NF-κB and the JAK-STAT cascades, which are known to exert fundamental roles in MM pathogenesis. CK2 blockade was associated to an accumulation of the inhibitor of NF-κB (IκBα) at baseline conditions as well as upon a strong NF-κB-activating stimulus, such as TNF-α. Consequently, the transcriptional activity of NF-κB was found substantially compromised. Moreover, CK2 inhibition led to an impaired phosphorylation of STAT3 on Tyr705 and Ser727. Growth stimuli, such as IGF-I and IL-6, were not able to overcome the lower cell survival frequency consequent to CK2 inhibition, suggesting a central role for this protein kinase downstream manifold signaling pathways. As a result, myeloma cells with less active CK2 were much more sensitive to the cytotoxic effect of a chemotherapeutic drug employed in MM, such as melphalan or the new-generation drug proteasome inhibitor bortezomib.
CK1α and CK2 in multiple myeloma: regulation of homeostatic/stress response pathways
It has been demonstrated in some cancer cells that also CK1, together with CK2 and GSK3β, takes part in the regulation of Hsp70 and Hsp90, influencing their binding to the co-chaperones HOP (Hsp70-Hsp90 organizing protein) and the chaperone-binding ubiquitin ligase CHIP to determine the cellular protein folding/degradation balance . The C-terminal phosphorylation of Hsp70 and Hsp90 in proliferating cancer cells enhances the assembly with HOP, increasing its protein folding activity. The non-phosphorylated chaperones preferentially bind to CHIP, mediating degradation of client proteins. It has been shown that melanoma, bladder, gastric, lung, breast, and pancreas cancer cells contain high levels of HOP, leading to high proliferation. Moreover, primary human breast tissue showed increased phosphorylated chaperones, compared to healthy samples. Even if a clear function of CK2 in UPR has been demonstrated in MM, given the role of CK1 on HOP and CHIP co-chaperones in other cancers, it is likely that also CK1α takes part in ER/stress, UPR in MM. Therefore, further research will have to clarify the exact role of CK1α in Hsp90-Hsp70 co-chaperone assembly and regulation in MM.
CK2 and CK1α role in bone marrow MM-stroma-delivered signals
Regarding CK1α, this kinase provided a growth advantage in MM plasma cellular clone evolution by impinging on pivotal signalling cascades important for MM cell survival. In particular, by inhibiting PCs and bone BM survival signaling molecules (like β-catenin and AKT), CK1α silencing/inhibition determined MM cell apoptosis, even when co-cultured with BMSCs, overcoming stromal cell protection . Moreover CK1α inhibition enhanced bortezomib and lenalidomide cytotoxicity on MM cells grown alone and/or on stromal cells, pointing to a function of CK1α on chemotherapy resistance. A recent study by Costa et al.  reported a role of CK1α in human dendritic cell (DC) maturation, modulating the mesenchymal stromal cell (MSC) inhibitory properties on DC evolution. CK1α silencing in MSC blunted the inhibitory effect of MSC on DC differentiation, increasing the expression of DC maturation markers (CD80, DC86, CD209) in a mechanism similar to that exerted by lenalidomide on DC of MM patients. This could suggest a potential role of CK1α in the modulation of MSC immunomodulatory properties. Further experiments would be necessary to prove this concept.
CK1α and CK2 inhibition as a rational therapeutic approach in MM
Protein kinases are rational therapeutic targets since they are druggable with small molecule inhibitors. The proof-of-concept of the effectiveness of anti-kinase therapy was first shown in chronic myeloid leukemia, in which the inhibitor imatinib mesylate has successfully targeted the aberrant kinase activity of BCR-ABL1 . Thereafter, a number of kinase inhibitors have entered the clinical arena. Recently, the B cell receptor signaling inhibitors idelalisib (which targets the PI3Kδ) and ibrutinib (which targets BTK) have shown an unprecedented clinical activity in B cell neoplasms [79, 80]. Nevertheless, in the anti-MM therapeutic armamentarium, kinase inhibitors are still lacking on the clinical ground.
The protein kinases CK1α and CK2 here discussed have demonstrated to play an important pro-survival role in MM. Thus, their inhibition could represent a rational approach in the therapy of this B cell malignancy. Recently, our group has shown that protein kinase CK2 sustains the growth of many blood cancers, including MM , MCL , DLBCL , and AML . Moreover, the CK2 inhibitor CX-4945 is now in phase I clinical trial in solid tumors and relapsed/refractory MM. Clinical-grade, oral ATP-competitive small molecule CK2 inhibitors have been described and tested in preliminary clinical trials in solid tumors and also MM, even though no results of these trials have been published. These already available drugs or other compounds in development could be tested in combination therapies with conventional or novel agents. Indeed, CK2 inhibitors boost chemotherapy toxicity in cultured MM  and other hematologic [83, 84, 85] and solid tumor cells . However, CK2 inhibition has proven to be a rational strategy also in combination with novel agents under study or already available in the therapy of MM. In particular, CK2 inhibition combined with the Hsp90 inhibitors geldanamycin or 17-AAG showed a remarkable in vitro and in vivo synergistic/cooperative cytotoxic activity in mouse models of MM . Moreover, the association of the proteasome inhibitor bortezomib with K27 or CX-4945 showed a synergistic cytotoxic effect on MM and MCL cells .
Since CK2 inhibition is associated with the impairment of NF-κB and STAT3 activation and signaling, a rational use of CK2 inhibitors would be together with NF-κB and STAT3 inhibitors. In support of this approach, data from our laboratory have demonstrated that CK2 inhibitors and STAT3 or IKK inhibitors may cooperate in inducing cell killing of AML blasts . If this holds true also in MM, which is a malignancy highly dependent on these two cascades, remains to be determined.
On the contrary, CK1α inhibition remains still an approach far from applicability in the clinical scenario, since no selective inhibitors targeting only this isoform of the CK1 family are available (D4476 is believed to be a dual CK1α/δ inhibitor). Nevertheless, the data here discussed support the idea that targeting CK1α might be beneficial at least in a subset of high-expressing MM patients. Moreover, it could always be possible to envision the dual CK1α/δ inhibition as an alternative approach, even if more data should be produced in this regard. Altogether, our in vitro studies indicate that CK1α inactivation may boost bortezomib and lenalidomide cytotoxicity. Further research should be pursued to confirm and extend these data.
In conclusion, CK1a and CK2 are two Ser/Thr kinases whose role in controlling signaling pathways involved in proliferation, survival and stress resistance in MM has been robustly established. Moreover, the results highlight the role of CK1α and CK2 in sustaining BMSCs-delivered growth clues to MM cells suggesting that these kinases could represent targets to disrupt MM cell intrinsic as well as extrinsic survival mechanisms. Even though most of the data on the role of CK1α and CK2 in MM have been generated in preclinical experimental models, the strategy of inhibition of these two kinases appears to lay on a strong rationale. It could be anticipated that the inhibition of CK1α and/or CK2 could cooperate or synergize with conventional or novel anti-MM agents. CK1α and CK2 might therefore be taken into consideration for future therapeutic strategies in the treatment of this hard-to-defeat malignancy.
This work was supported by a grant from the Associazione Italiana per la Ricerca sul Cancro (AIRC) to FP (IG 14481 and IG 18387). SM was supported by a Fondazione Umberto Veronesi fellowship.
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